JP7490035B2 - In vitro differentiation of pluripotent stem cells into pancreatic endoderm cells (PECs) and endocrine cells - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/781,005, filed March 14, 2013, and U.S. Utility Application No. 14/106,330, filed December 13, 2013, the disclosures of which are incorporated herein in their entireties.

The present invention relates generally to the isolation, maintenance, and use of cell cultures. More specifically,
The present invention relates to the differentiation of pluripotent stem cells into pancreatic endoderm cells ( ), which are enriched for a non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-), but relatively enriched for cells committed to the endocrine lineage (CHGA+).
The present invention relates to cell compositions and methods for the in vitro differentiation of pluripotent stem cells into mammalian (PEC) cultures.
Also described herein are cell compositions and methods for the in vitro differentiation of pluripotent stem cells into endocrine cells that produce insulin in response to glucose stimulation in vitro. Also described herein are cell compositions and methods for the in vitro differentiation of pluripotent stem cells into endocrine cells that produce insulin in vitro.

The development of expanded populations of human pancreatic β-cells that can be used for cell transplantation is a major goal in diabetes research. In recent years, attention has focused on the use of pluripotent stem cells as a potential renewable source of β-cells. The nature of pancreatic endocrine differentiation is complex and currently remains to be elucidated, and differentiation of pluripotent stem cells into mature and functional β-cells has not yet been demonstrated in vitro.
To date, only progenitor cells such as pancreatic endoderm cells (PECs) have been transplanted into rodents, demonstrating human-specific β-cell function after 8-12 weeks. PEC cell populations contain at least two cell populations: a non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-) and cells committed to the endocrine lineage (CHGA+).
It was discovered that a subpopulation of non-endocrine multipotent pancreatic progenitors (CHGA-) matures and forms β cells in vivo when implanted in vitro.
A long maturation time in vivo is required (8-12 weeks), which is
This could be shortened or eliminated if the in vitro PEC population contains more cell types that mature into β cells in vivo (i.e., non-endocrine multipotent pancreatic progenitors (CHGA-)), (b) if terminal (β cell) differentiation is achieved in vitro, or (c) if an in vitro precursor population that is further along the differentiation pathway than currently used, i.e., a developmentally more advanced cell population, is identified.

Without limiting the present application to any one theory, it is believed that pluripotent stem cells are in vivo
A possible explanation for the failure of β-cells to mature into fully functional cells in vitro is that
In vitro derived cells do not have expression of the same markers at the same time points as in vivo mammalian pancreatic development. For example, NGN3 expression during in vitro differentiation precedes in vivo mammalian pancreatic development.
Indeed, in conventional four-stage differentiation protocols described in Applicant's numerous publications and patents, which are incorporated herein by reference in their entireties, PEC populations have been observed to express NGN3 and KX2.2. Thus, suppressing or inhibiting NGN3 expression until after differentiation of a PDX1/NKX6.1 co-positive non-endocrine multipotent pancreatic progenitor subpopulation has been demonstrated to inhibit the in vitro differentiation of pluripotent stem cells into mature and functional beta cells or cells committed to the endocrine lineage (C
This represents an important step in achieving differentiation of a PEC population that is enriched for non-endocrine multipotent pancreatic progenitor cells (CHGA-) compared to HGA+.

Identifying protocols that delay expression of NGN3 and NKX2.2 is not trivial because the differentiation protocol results in the differentiation of a non-endocrine multipotent pancreatic progenitor subpopulation (C
HGA-) formation should not be perturbed, and preferably should be increased, and cell loss should be minimized, or at a minimum adequate cell mass and cell yield (i.e., micrograms, grams, kilograms of cells) should be maintained.

Furthermore, because NGN3 expression is required for the initiation of endocrine cell differentiation, promoting islet cell maturation, and maintaining islet function, stimulating or inducing NGN3 expression in cells committed to the endocrine lineage (CHGA+) may promote the generation of mature and functional β-cells from pluripotent stem cells in vitro.
It is a key step in achieving differentiation of cells.

In one aspect, an in vitro method for generating a cell population comprising human PDX1-positive pancreatic endoderm cells is provided.
The method includes the step of culturing definitive endoderm.
A cell population containing TGFβ superfamily growth factors and W
In some embodiments, the TGFβ superfamily growth factor is selected from the group consisting of Nodal, activin A, activin B, BMP2, BMP4, GDF8, and the like, thereby generating a cell population comprising PDX1-positive pancreatic endoderm cells.
, GDF-10, GDF-11 and GDF-15. In some embodiments, the method further comprises contacting the cell population comprising definitive endoderm lineage cells with an ERBB receptor kinase activating agent. In some embodiments, the TGFβ superfamily growth factor is activin A. In some embodiments, the Wnt family member is Wnt3a. In some embodiments, the ERBB receptor kinase activating agent is heregulin.

In some embodiments, the cell population comprising PDX1-positive pancreatic endoderm cells is cultured at least 25 nM.
In some embodiments, the cells are contacted with a TGFβ superfamily growth factor at 100 μg/mL.
The cell population comprising PDX1-positive pancreatic endoderm cells is contacted with at least 50 ng/mL of a TGFβ superfamily growth factor. In some embodiments, the cell population comprising PDX1-positive pancreatic endoderm cells is contacted with at least 75 ng/mL of a TGFβ superfamily growth factor.

In some embodiments, the cell population comprising PDX1-positive pancreatic endoderm cells is cultured at least 25 nM.
In some embodiments, the cell population comprising PDX1-positive pancreatic endoderm cells is contacted with at least 50 ng/mL of a Wnt family member. In some embodiments, the cell population comprising PDX1-positive pancreatic endoderm cells is contacted with at least 75 ng/mL of a Wnt family member.

In some embodiments, the cell population is contacted with 5-15 times less of an ERBB receptor kinase activating agent than each of a TGFβ superfamily growth factor and a Wnt family member. In some embodiments, at least about 50% of the cell population comprising PDX1-positive pancreatic endoderm cells are PDX1-positive pancreatic endoderm cells. In some embodiments,
At least about 60% of a cell population comprising PDX1-positive pancreatic endoderm cells are PDX1-positive pancreatic endoderm cells. In some embodiments, at least about 70% of a cell population comprising PDX1-positive pancreatic endoderm cells are PDX1-positive pancreatic endoderm cells. In some embodiments, PDX1
At least about 80% of a cell population comprising PDX1-positive pancreatic endoderm cells are PDX1-positive pancreatic endoderm cells. In some embodiments, at least about 90% of a cell population comprising PDX1-positive pancreatic endoderm cells are PDX1-positive pancreatic endoderm cells.

In embodiments, the PDX1-positive pancreatic endoderm cells further express NKX6.1;
In some embodiments, the PDX1-positive pancreatic endoderm cells are
In some embodiments, PDX1-positive pancreatic endoderm cells do not express NKX6.HGA.
1 and PFT1A, and substantially no expression of NGN3. In some embodiments, PDX1-positive pancreatic endoderm cells are multipotent cells that can differentiate into unipotent immature beta cells.

In one aspect, a human pancreatic endoderm cell culture is provided comprising definitive endoderm lineage cells and a TGFβ superfamily growth factor and a Wnt family member. In embodiments, the TGFβ superfamily growth factor is selected from the group consisting of Nodal, activin A, activin B, BMP2, BMP4, GDF8, GDF-10, GDF-11, and GDF-15.
In some embodiments, the method further comprises contacting the cell population comprising definitive endoderm lineage cells with an ERBB receptor kinase activating agent. In some embodiments, the TGFβ superfamily growth factor is activin A. In some embodiments, the Wnt family member is Wnt3a. In some embodiments, the ERBB receptor kinase activating agent is heregulin.

In another aspect, in vitro unipotent human immature beta cells are provided. In some embodiments, the unipotent human immature beta cells express INS and NKX6.1 and NGN.
In some embodiments, the unipotent human immature beta cells do not substantially express IL-1, IL-2, or IL-3. In some embodiments, the unipotent human immature beta cells are capable of differentiating into mature beta cells. In some embodiments, the differentiation is in vivo. In some embodiments, the unipotent human immature beta cells form part of a cell population. In some embodiments, at least 10% of the cell population are immature beta cells.
In embodiments, at least 20% of the cell population are immature beta cells. In embodiments, at least 30% of the cell population are immature beta cells. In embodiments, at least 40% of the cell population are immature beta cells. In embodiments, at least 50% of the cell population are immature beta cells. In embodiments, at least 60% of the cell population are immature beta cells. In embodiments, at least 80% of the cell population are immature beta cells. In embodiments, at least 90% of the cell population are immature beta cells. In embodiments, at least 98% of the cell population are immature beta cells. In embodiments, the human immature beta cells are monohormonal (sin
gly hormonal). In embodiments, the unipotent human cells express MAFB.

In one aspect, a method for generating unipotent human immature beta cells is provided, the method comprising:
The method includes contacting human definitive endoderm lineage cells in vitro with a TGFβ superfamily member and a Wnt family member, thereby generating immature beta cells. In some embodiments, the unipotent human immature beta cells are INS, NKX6.
In some embodiments, the TGFβ superfamily member expresses Nodal, activin A, activin B, BMP2, BMP4,
It is selected from the group consisting of GDF8, GDF-10, GDF-11 and GDF-15.
In some embodiments, the method comprises transfecting human definitive endoderm lineage cells in vitro with ERB.
The method further comprises contacting the TGFβ receptor kinase activating agent with a TGFβ superfamily growth factor. In some embodiments, the TGFβ superfamily growth factor is activin A. In some embodiments, the
The Wnt family member is Wnt3a. In embodiments, the ERBB receptor kinase activating agent is heregulin. In embodiments, the TGFβ superfamily growth factor is a BMP.

In some embodiments, the method further comprises contacting the human definitive endoderm lineage cells in vitro with nicotinamide, retinoic acid or a retinoic acid analog, a rho kinase inhibitor, or a gamma sequestrantase inhibitor. In some embodiments, the retinoic acid is selected from the group consisting of all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-retinoic acid), and the like.
In some embodiments, the retinoic acid is selected from the group consisting of all-trans retinoic acid (RA). In some embodiments, the retinoic acid is 13-cis-retinoic acid (13-cis-RA). In some embodiments, the retinoic acid is arotinoic acid (TTNPB). In some embodiments, the rho kinase inhibitor is selected from the group consisting of Y-27632, fasudil, H-1152P, Wf
In some embodiments, the rho is selected from the group consisting of rho-536 and Y-30141.
The kinase inhibitor is Y-27632.

In some embodiments, the gamma selenase inhibitor is gamma selenase inhibitor I (G
Gamma-secretase inhibitor II (GSI II), Gamma-secretase inhibitor III (GSI III), Gamma-secretase inhibitor IV (GSI IV), Gamma-secretase inhibitor V (GSI V), Gamma-secretase inhibitor VI (GSI VI)
, gamma-secretase inhibitor VII (GSI VII), gamma-secretase inhibitor IX
(GSI IX), (DAPT), gamma-secretase inhibitor XI (GSI XI), gamma-secretase inhibitor XII, (GSI XII), gamma-secretase inhibitor XII
I (GSI XIII), gamma-secretase inhibitor XIV (GSI XIV), gamma-secretase inhibitor XVI (GSI XVI), gamma-secretase inhibitor XVII (G
Gamma-secretase inhibitor XIX (GSI XIX), gamma-secretase inhibitor XX (GSI XX), gamma-secretase inhibitor XXI (GSI XXI
), gamma 40 secretase inhibitor I, gamma 40 secretase inhibitor II and RO
In some embodiments, the gamma selenase inhibitor is selected from the group consisting of RO4929097. In some embodiments, the gamma selenase inhibitor is G
It is SI IV.

In another aspect, a method for generating mature beta cells in vivo is provided.
The method comprises: (a) contacting human definitive endoderm lineage cells in vitro with a TGFβ superfamily member and a Wnt family member, thereby generating immature beta cells; and (b) transplanting the immature beta cells of step (a) into a mammalian subject, and differentiating the immature beta cells in vivo to generate a population of cells comprising mature beta cells. In some embodiments, the method further comprises causing the mature beta cells to produce insulin in response to glucose stimulation. In some embodiments, the TGFβ superfamily growth factor is selected from the group consisting of Nodal, activin A and activin B, GDF-8, GDF-11, and GDF-15. In some embodiments, the method further comprises contacting the human definitive endoderm lineage cells in vitro with an ERBB receptor kinase activating agent. In some embodiments, the TGFβ superfamily growth factor is activin A. In some embodiments, the Wnt family member is Wnt3a. In some embodiments, the ERBB receptor kinase activating agent is heregulin.

In some embodiments, human definitive endoderm lineage cells are cultured in a culture medium containing at least 25 ng/mL TGFβ.
In some embodiments, the human definitive endoderm lineage cells are contacted with at least 50 ng/mL of a TGFβ superfamily growth factor. In some embodiments, the human definitive endoderm lineage cells are contacted with at least 75 ng/mL of a TGFβ superfamily growth factor.

In some embodiments, the human definitive endoderm lineage cells are contacted with at least 25 ng/mL of a Wnt family member. In some embodiments, the human definitive endoderm lineage cells are contacted with at least 50 ng/mL of a Wnt family member. In some embodiments, the human definitive endoderm lineage cells are contacted with at least 75 ng/mL of a Wnt family member.

In embodiments, human definitive endoderm lineage cells are contacted with a TGFβ superfamily growth factor and an ERBB receptor kinase activating agent that is 5-15 times less than Wnt family members. In embodiments, at least 10% of the cell population are immature beta cells. In embodiments, at least 20% of the cell population are immature beta cells. In embodiments, at least 30% of the cell population are immature beta cells.
In some embodiments, at least 40% of the cell population are immature beta cells. In some embodiments, at least 50% of the cell population are immature beta cells. In some embodiments, at least 60% of the cell population are immature beta cells. In some embodiments, at least 70% of the cell population are immature beta cells. In some embodiments, at least 80% of the cell population are immature beta cells. In some embodiments, at least 90% of the cell population are immature beta cells. In some embodiments, the immature beta cells express INS and NKX6.1 and do not substantially express NGN3. In some embodiments, the immature beta cells express INS, NKX6.1 and MAFB and do not substantially express NGN3.

In one aspect, in vitro unipotent human immature beta cells are provided. In some embodiments, the unipotent human immature beta cells express INS and NKX6.1, and NGN3.
In some embodiments, the unipotent human immature beta cells are capable of maturation into mature beta cells.

In one aspect, a method is provided for generating mature beta cells in vivo, comprising: a. contacting human definitive endoderm lineage cells in vitro with a TGFβ superfamily member and a Wnt family member, thereby generating immature beta cells, b. transplanting the immature beta cells of step (a) into a mammalian subject, and c. maturing the immature beta cells in vivo to generate a population of cells comprising insulin-secreting beta cells.

In one aspect, unipotent human immature beta cells are provided that express INS and NKX6.1 and do not substantially express NGN3. In some embodiments, the unipotent human immature beta cells are capable of maturing into mature beta cells. In some embodiments, the unipotent human immature beta cells further express MAFB.

In one aspect, an in vivo mouse model expressing INS and NKX6.1 and not substantially expressing NGN3
In vitro unipotent human immature beta cells are provided. In some embodiments, the unipotent human immature beta cells are capable of maturing into mature beta cells. In some embodiments, the unipotent human immature beta cells further express MAFB.

In one aspect, a method for generating unipotent human immature beta cells is provided, the method comprising:
The method includes contacting human definitive endoderm lineage cells in vitro with a TGFβ superfamily member and a Wnt family member, thereby generating unipotent human immature beta cells. In some embodiments, the unipotent human immature beta cells are INS, N
In some embodiments, the method further comprises contacting the human definitive endoderm lineage cells in vitro with an ERBB receptor kinase activating agent. In some embodiments, the TGFβ superfamily growth factor is activin A. In some embodiments, the Wnt family member is Wn
t3a. In several embodiments, the ERBB receptor kinase activating agent is heregulin.

In one aspect, a method is provided for generating mature beta cells in vivo, the method comprising: a. contacting human definitive endoderm-lineage cells in vitro with a TGFβ superfamily member and a Wnt family member, thereby generating immature beta cells; b. transplanting the immature beta cells of step (a) into a mammalian subject; and c. maturing the immature beta cells in vivo to generate a population of cells comprising insulin-secreting beta cells. In embodiments, the method further comprises contacting the human definitive endoderm-lineage cells in vitro with an ERBB receptor kinase activating agent. In embodiments, the TGFβ superfamily growth factor is activin A. In embodiments, the Wnt family member is Wnt3a. In embodiments, the ERBB receptor kinase activating agent is heregulin. In embodiments, the human definitive endoderm-lineage cells are transplanted into a mammalian subject with at least 50 ng/mL of TGFβ superfamily member and a Wnt family member, thereby generating immature beta cells.
In some embodiments, the human definitive endoderm lineage cells are contacted with at least 25 ng/mL of a TGFβ superfamily growth factor. In some embodiments, the human definitive endoderm lineage cells are contacted with at least 25 ng/mL of a Wnt family member. In some embodiments, the human definitive endoderm lineage cells are contacted with a TGFβ superfamily growth factor and 5-15 times less of an ERBB receptor kinase activating agent than a Wnt family member. In some embodiments, the immature beta cells are contacted with INS and NK
X6.1 and does not substantially express NGN3. In some embodiments, immature beta cells express INS, NKX6.1 and MAFB and do not substantially express NGN3.

In one embodiment, compositions and methods are provided for differentiating pluripotent stem cells or progenitor cells derived from pluripotent stem cells into endocrine and non-endocrine populations. In one aspect, endocrine cells express at least CHGA (or CHGA+) and non-endocrine cells express CHGA.
In one embodiment, these cell populations do not express GA (or CHGA-).
In another embodiment, the endocrine and non-endocrine subpopulations may be multipotent progenitor/precursor subpopulations, such as non-endocrine multipotent pancreatic progenitor subpopulations or endocrine multipotent pancreatic progenitor subpopulations, or may be unipotent subpopulations, such as immature endocrine cells, preferably immature beta cells, immature glucagon cells, etc.

In one embodiment, compositions and methods are provided for in vitro differentiation of pluripotent stem cells into pancreatic endoderm cell (PEC) cultures that contain both endocrine and non-endocrine subpopulations, where the expression of NGN3 is suppressed. In one aspect, the expression of NKX2.2 is also suppressed in the PEC cultures. In one aspect, the expression of CHGA is also suppressed in the PEC cultures. In one aspect, the expression of PDX1 is also suppressed in the PEC cultures.
and NKX6.1 expression is co-expressed in a single cell in the PEC culture. In one aspect, less than 50%, preferably less than 40%, 30%, more preferably less than 20%, 10%, 5%, 2%, 1% of the cells in the PEC culture express NGN3, NKX2.2 or CHGA. In one aspect, the PEC culture is enriched for a non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-). In one aspect, the PEC culture expresses markers of the non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-), such as PDX1 and/or NKX6.1. In one aspect, the PEC culture with enriched non-endocrine multipotent progenitor subpopulation (CHGA-) is implanted into a mammalian host and the graft is allowed to mature and produce insulin in response to glucose in vivo. In one aspect, PEC cultures with an enriched non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-) mature at a faster rate in vivo than PEC cultures without an enriched non-endocrine multipotent progenitor subpopulation (CHGA-). In one aspect, implanted PEC cultures with an enriched non-endocrine multipotent progenitor subpopulation (CHGA-) have a higher in vivo function as measured by C-peptide (insulin) production in response to glucose stimulation than implanted PEC cultures without an enriched non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-) at about 10 weeks or about 12 weeks post-implantation.
There is at least a two-fold improvement over EC cultures.

In one embodiment, compositions and methods for differentiating pluripotent human stem cells in vitro are provided, in which expression of genes typical of endocrine differentiation and development, such as NGN3 and NKX2.2, is associated with non-endocrine multipotent pancreatic progenitors (CHGA- and PDX1/NKX6
Compositions and methods are provided in which expression of the .1 gene (.1 co-expression) is inhibited until post-production.

In one embodiment, a method for differentiating pluripotent stem cells into PEC cultures in vitro comprises the steps of: (a) obtaining a population of PDX1-negative foregut endoderm cells;
and b) contacting the cell population of step (a) with an agent that activates a TGFβ receptor family member, thereby resulting in a cell culture comprising PECs that do not substantially express a marker selected from the group comprising CHGA, NGN3, or NKX2.2. In one aspect, the PECs comprise a subpopulation of cells that co-express PDX1 and NKX6.1. In one aspect, the PECs are non-endocrine multipotent pancreatic progenitors (CHGA, NGN3, or NKX2.2).
In one embodiment, the agent that activates a TGFβ receptor family member is selected from the group consisting of Nodal, activin A, activin B, BMP2, BMP4, GDF8, and the like.
In one embodiment, activin A is administered at 100 ng/mL, 75 ng/mL, 50 ng/mL, or mixtures thereof at stage 3.
ng/mL, 25 ng/mL or 10 ng/mL, and at stage 4 at 15 ng/mL
In one embodiment, activin A is added at PDX
PEC cultures formed by adding to a population of C-negative foregut endoderm cells have at least two-fold improved in vivo function, as measured by C-peptide (insulin) production in response to glucose stimulation, compared to PEC cultures without an enriched non-endocrine multipotent progenitor subpopulation (CHGA-).

In one aspect, PEC cultures with enriched non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-) have at least two-fold improved in vivo function as measured by insulin production in response to glucose stimulation compared to PEC cell cultures without enriched non-endocrine multipotent progenitor subpopulation (CHGA-).

In one embodiment, a method is provided for in vitro differentiation of pluripotent stem cells into a cell population comprising PECs, the method comprising the steps of: (a) obtaining a population of PDX1-negative foregut endoderm cells; and (b) contacting the cell population of step (a) with an agent that activates a TGFβ receptor family member, thereby resulting in a PEC population that does not substantially express a marker selected from the group comprising CHGA, NGN3 or NKX2.2. In one aspect, the PEC population comprises cells that co-express PDX1 and NKX6.1. In one aspect, the PEC population comprises non-endocrine multipotent pancreatic progenitor cells (CHGA-).
In one aspect, the agent that activates a TGFβ receptor family member is No.
dal, activin A, activin B, BMP2, BMP4, GDF8, GDF-10,
In one embodiment, the antibody is selected from the group consisting of GDF-11, GDF-15 and mixtures thereof.
The agent that activates TGFβ receptor family members is activin A. In one embodiment, activin A is administered at 100 ng/mL, 75 ng/mL, 50 ng/mL, or 100 ng/mL at stage 3.
, 25 ng/mL or 10 ng/mL, and in stage 4, 15 ng/mL, 10 ng/mL
In one embodiment, the EGF family ligand is heregulin (also known as neuregulin 1 (NRG1)), neuregulin 2 (NRG2), or 5ng/mL.
In one embodiment, the EGF family ligand is heregulin. In one embodiment, heregulin is administered at 2 ng/mL, 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL, 100 ng/mL, 15 ng/mL, 100 ng/mL, 15 ng/mL, 20 ng/mL, 100 ng/mL, 25 ng/mL, 15 ng/mL, 20 ng/mL, 15 ng/mL, 25 ng/mL, 1 ...
L, 10ng/mL, 20ng/mL, 15ng/mL, 30ng/mL, 40ng/mL
In one embodiment, PEC cultures formed by adding activin A and heregulin to a PDX1-negative foregut endoderm cell population are added at 50 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL or 80 ng/mL. ...
In vivo function, as measured by insulin) production, is improved by at least 2-fold over PEC cultures without an enriched non-endocrine multipotent progenitor subpopulation (CHGA-).

In one embodiment, a method for in vitro differentiation of pluripotent stem cells into a cell culture comprising PECs, comprising: (a) obtaining a population of PDX1-negative foregut endoderm cells; and (b) contacting the cell population of step (a) with at least an agent that activates a TGFβ receptor family member, an EGF family ligand, or an agent that activates the Wnt signaling pathway, either alone or in combination, thereby producing a differentiation medium comprising:
PE that does not substantially express a marker selected from the group including NGN3 or NKX2.2
and generating a cell culture comprising PE.
The PEC culture comprises cells that co-express PDX1 and NKX6.1. In one aspect, the PEC cell culture comprises a non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-).
Agents that activate GFβ receptor family members include Nodal, activin A,
Activin B, BMP2, BMP4, GDF8, GDF-10, GDF-11, GDF-
15 and mixtures thereof. In one aspect, the agent that activates a TGFβ receptor family member is activin A. In one aspect, the agent that activates the Wnt signaling pathway is WNT1, WNT2, WNT2B, WNT3, WNT3
A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8
A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11,
The Wnt family member is selected from the group including WNT16 and Wnt3A.
In one aspect, the Wnt family member is Wnt3A. In one aspect, activin A
was added at 100 ng/mL, 75 ng/mL, 50 ng/mL, 25 ng/mL or 10 ng/mL in stage 3, and at 15 ng/mL, 10 ng/mL or 5 ng/mL in stage 4.
In one embodiment, heregulin is added at 2 ng/mL, 5 ng/mL, 10 ng/mL,
/mL, 20ng/mL, 15ng/mL, 30ng/mL, 40ng/mL, 50ng
In one embodiment, WNTs are added at 5 ng/mL, 25 ng/mL, 50 ng/mL to 75 ng/mL, more preferably 50 ng/mL.

In one embodiment, activin A is added alone or in combination with WNT at stage 3. In another embodiment, activin A is added together with WNT or WNT and heregulin at stage 3. In yet another embodiment, activin A is added alone or in combination with heregulin at stage 4.

In one aspect, more than 10%, preferably more than 20%, more than 30%, more than 40%, more preferably more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 10%, more preferably more than 20%, more preferably more than 30%, more preferably more than 40%, more preferably more than 50%, more than 60%, more than 70%, more than 8
In one aspect, PEC cell cultures formed by adding activin A, heregulin and WNT to a PDX1-negative foregut endoderm cell population exhibit an enriched non-endocrine multipotent progenitor subpopulation (CHGA-) that is functional in vivo as measured by insulin production in response to glucose stimulation.
The improvement is at least two-fold over PEC cell cultures without CHGA-.

In one embodiment, compositions and methods are provided for in vitro differentiation of pluripotent stem cells into PEC cultures comprising minimally committed cells (CHGA+). In one aspect, the endocrine committed cells are characterized by expression of CHGA.
In one embodiment, less than 50%, preferably less than 40%, less than 30% of the cells in the PEC culture
More preferably, less than 20%, less than 10%, less than 5%, less than 2%, less than 1% are cells committed to the endocrine lineage (CHGA+).

In one embodiment, compositions and methods are provided for in vitro differentiation of pluripotent stem cells into substantial PEC cultures and further differentiation of the PEC cultures into endocrine cells in vitro. In one aspect, the endocrine cells express CHGA. In one aspect, the endocrine cells are capable of producing insulin in vitro. In one aspect, the in vitro endocrine insulin-secreting cells are capable of producing insulin in response to glucose stimulation. In one aspect, more than 10%, preferably more than 20%, more than 30%, more than 40%, more preferably more than 50%, more than 60%, more than 70%, more than 80% of the cells in the cell population express CHGA.
greater than, greater than 90%, greater than 95%, greater than 98% or 100% are endocrine cells.

In one embodiment, a method is provided for differentiating pluripotent stem cells into endocrine cells in vitro, comprising: (a) obtaining a cell population comprising PECs; and (b) contacting the cell population of step (a) with a growth factor selected from the group consisting of bone morphogenetic proteins (BMPs), hedgehog inhibitors, platelet derived growth factor (PDGF), members of the fibroblast growth factor family, retinoids, insulin growth factors 1 and 2 (IGF1 and IGF2), and nicotinamide, thereby resulting in endocrine cells. In one aspect, the endocrine cells express CHGA. In one aspect, the endocrine cells are immature insulin-producing beta cells in vitro. In one aspect, the endocrine cells are mature, loaded into an implantable semipermeable encapsulation device,
In one embodiment, more than 10%, preferably more than 20%, more than 30%, more than 40% of the cells in the cell population are capable of producing insulin in response to glucose stimulation in vivo.
More preferably, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 9
More than 8% or 100% are endocrine cells.
In one embodiment, PDGF is added at 10 ng/mL to 100 ng/mL. In one embodiment, BMP is added at 10 ng/mL to 20 ng/mL. In one embodiment, FGF2 is added at 2 ng/mL to 20 ng/mL. In one embodiment, IGF1 or IGF2 is added at 25 ng/mL to 100 ng/mL. In one embodiment, the above compounds are added in combination with each other. In one embodiment, the member of the fibroblast growth factor family is FGF2. In one embodiment, the retinoid is retinoic acid and/or a retinoic acid analog such as TTNPB or TT3, insulin-like growth factor I (IGF-I or IGF1) and insulin-like growth factor-II (IGF-II or IGF2). In one embodiment, the bone morphogenetic protein is BMP4. In one aspect, the hedgehog inhibitor is sonic hedgehog, KAAD-cyclopamine, KAAD-cyclopamine analogs, jervine, jervine analogs, hedgehog pathway blocking antibodies and any other inhibitor of hedgehog pathway function known to one of skill in the art.

In one embodiment, a method for in vitro differentiation of pluripotent stem cells into endocrine cells comprises: (a) obtaining a cell population comprising PECs; and (b) in vitro differentiation of pluripotent stem cells into endocrine cells.
and contacting in vitro the cell population of step (a) with a gamma secretase inhibitor, thereby generating endocrine cells.
The endocrine cells express CHGA. In one aspect, the endocrine cells are immature insulin-producing beta cells in vitro. In one aspect, the endocrine cells are immature insulin-producing beta cells in vitro.
In one aspect, the endocrine cells are mature, capable of producing insulin in response to glucose stimulation in vivo, loaded into an implantable semipermeable encapsulation device. In one aspect, the gamma secretase inhibitor is N-[N-(3,5-diflurophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), R
O44929097, 1-(S)-endo-N-(1,3,3)-trimethylbicyclo[2.2.1]hept-2-yl)-4-fluorophenylsulfonamide, WPE-I
II31C, S-3-[N'-(3,5-difluorophenyl-α-hydroxyacetyl)-L-alanyl]amino-2,3-dihydro-1-methyl-5-phenyl-1H-1
, 4-benzodiazepin-2-one, (N)-[(S)-2-hydroxy-3-methyl-
butyryl]-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,
7-Tetrahydro-2H-3-benzazepin-2-one, BMS-708163 (Av
agacestat), BMS-708163, Semagacestat (LY450
139), Semagacestat (LY450139), MK-0752, MK-0
752, YO-01027, YO-01027 (dibenzazepine, DBZ), LY-41
1575, LY-411575, or LY2811376. In some embodiments, the gamma sequestrant is present in the cell culture or cell population at a concentration of about 0.
In a preferred embodiment, the gamma selenase inhibitor is present in the cell culture or cell population at a concentration ranging from about 0.1 μM to about 100 μM.
In one embodiment, the gamma secreta segregase inhibitor is removed after addition.

In embodiments described herein, compositions and methods are provided for differentiating pluripotent human stem cells into endocrine cells in vitro. In one aspect, the endocrine cells express CHGA. In one aspect, the endocrine cells are capable of producing insulin in vitro. In one aspect, the endocrine cells are immature endocrine cells, such as immature beta cells. In one aspect, the in vitro insulin-producing cells are capable of producing insulin in response to glucose stimulation.

In one embodiment, a method for producing insulin in vivo in a mammal comprises the steps of: (a) loading an implantable semipermeable device with a population of endocrine cells; (b) implanting the device with the endocrine cells into a mammalian host; and (c)
and maturing the endocrine cell population in said device in vivo, wherein at least a portion of the endocrine cells are insulin-secreting cells that produce insulin in response to glucose stimulation in vivo, thereby producing insulin in vivo in a mammal. In one aspect, the endocrine cells are derived from a cell composition that includes PECs with a larger subpopulation of non-endocrine multipotent pancreatic progenitors (CHGA-).
In another aspect, the endocrine cells are derived from a cell composition comprising PECs that are enriched in an endocrine subpopulation (CHGA+).In another aspect, the endocrine cells are immature endocrine cells, preferably immature beta cells.

In one aspect, endocrine cells are characterized as expressing PDX1 in vitro as compared to a PDX-1 positive pancreatic endoderm population or a non-endocrine (CHGA-) subpopulation that is PDX1/NKX6.1 positive.
and NKX6.1. In one aspect, the endocrine cells are generated from pluripotent stem cells that express relatively more PDX1 and NKX6.1 in vitro than the PEC non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-).
In one embodiment, bone morphogenetic proteins (BMPs) and retinoic acid (RA) analogs, either alone or in combination, are added to cell cultures to increase PDX1 and NKX6.1 expression.
The endocrine cells are increased relative to the EC non-endocrine multipotent progenitor subpopulation (CHGA-). In one aspect, the BMP is selected from the group including BMP2, BMP5, BMP6, BMP7, BMP8 and BMP4, more preferably BMP4. In one aspect, the retinoic acid analog is selected from the group including all-trans retinoic acid and TTNPB (4-[(E)-2
-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid arotinoic acid), or 0.1 to 10 μM AM-58
0(4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid), more preferably TTNPB.

In one embodiment, a method is provided for in vitro differentiation of pluripotent stem cells into endocrine cells and immature endocrine cells, preferably immature beta cells, comprising dissociating and reassociating aggregates. In one aspect, the dissociation and reassociation comprises:
This occurs at stage 1, stage 2, stage 3, stage 4, stage 5, stage 6, or stage 7, or a combination thereof. In one aspect, definitive endoderm, PDX1-negative foregut endoderm, PDX1-positive foregut endoderm, PEC, and/or endocrine cells and endocrine precursor/precursor cells are dissociated and reassociated. In one aspect, at stage 7, endocrine (C
The cell aggregates, which are comprised of a smaller non-endocrine (CHGA-) subpopulation compared to the HGA+ subpopulation, are dissociated and reaggregated. In one embodiment, more than 10%, preferably 20%, of the cells in the cell population
More than 30%, more than 40%, more preferably more than 50%, more than 60%, more than 70%, more than 80%, more than 9
Greater than 0%, greater than 95%, greater than 98% or 100% are endocrine (CHGA+) cells.

In one embodiment, endocrine cells generated during stage 4 PEC generation are removed, thereby generating non-endocrine multipotent pancreatic progenitors (CHGA-
) The enrichment of the subpopulation provides a method for differentiating pluripotent stem cells into endocrine cells in vitro.

In one embodiment, about 0.02% to 2%, preferably about 0.02% to 0.05%, preferably about 0.05% to 1%, of stage 6 and stage 7 cells as defined in Table 17 are
In one embodiment, a method is provided for differentiating pluripotent stem cells into endocrine cells in vitro by culturing them in low levels of Matrigel, preferably 0.05%.

In one embodiment, a method is provided for improving cell adhesion between PEC and endocrine cell aggregates by treating the cell culture with Matrigel and a rho-kinase or ROCK inhibitor. In one aspect, the rho-kinase or ROCK inhibitor is Y-27632.

In one embodiment, PEC cultures enriched for non-endocrine multipotent progenitor subpopulations (CHGA-) are generated by not adding Noggin family members at stage 3 and/or stage 4. In one embodiment, endocrine lineage-committed cells (
A PEC culture relatively enriched in CHGA+ is generated by not adding Noggin family members to stage 3 and/or stage 4. In one aspect, Noggin family members include Noggin, Chordin, Follistatin, Follistatin-like protein,
The compound is selected from the group including Cerberus, Coco, Dan, Gremlin, Sclerostin, PRDC (protein related to Dan and Cerberus).

In one embodiment, endocrine cells in culture are grown in medium containing high levels of exogenous glucose, where the exogenous glucose added is between about 1 mM and 25 mM, between about 1 mM and 20 mM, between about 5 mM and 6 mM,
Methods for maintaining the cells by culturing in a medium that is about 5 mM to 15 mM, about 5 mM to 10 mM, about 5 mM to 8 mM, in one aspect, the medium is a DMEM, CMRL or RPMI based medium.

In one embodiment, a method is provided for in vitro differentiation of pluripotent stem cells into endocrine cells, with and without dissociation and reassociation of cell aggregates. In one aspect, in vivo differentiation of endocrine cells is performed at stage 6 and/or stage 7.
The undissociated or dissociated and reassociated cell aggregates are cryopreserved or frozen without affecting the function of the endocrine cell. In one embodiment, the cryopreserved endocrine cell culture is thawed and cultured to
Once implanted, it functions in vivo.

In another embodiment, a culture system for differentiating pluripotent stem cells into endocrine cells is provided, comprising at least an agent capable of suppressing or inhibiting endocrine gene expression early in differentiation and an agent capable of inducing endocrine gene expression late in differentiation. In one aspect, an agent capable of suppressing or inhibiting endocrine gene expression is added to a culture consisting of pancreatic PDX1-negative foregut cells. In one aspect, an agent capable of inducing endocrine gene expression is added to a culture consisting of PDX1-positive pancreatic endoderm precursors or PECs. In one aspect, the agent capable of suppressing or inhibiting endocrine gene expression is an agent that activates the TGF-beta receptor family, preferably activin, with high levels of activin followed by low levels of activin. In one aspect, the agent capable of induc ...
,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), RO44929097, DAPT (N--[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester), 1-
(S)-endo-N-(1,3,3)-trimethylbicyclo[2.2.1]hepta-2
-yl)-4-fluorophenylsulfonamide, WPE-III31C, S-3-[N
'-(3,5-difluorophenyl-α-hydroxyacetyl)-L-alaninyl]amino-2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepine-
2-one, (N)-[(S)-2-hydroxy-3-methyl-butyryl]-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-
3-Benzazepin-2-one, BMS-708163 (Avagacestat), B
MS-708163, Semagacestat (LY450139), Semagac
estat (LY450139), MK-0752, MK-0752, YO-01027
, YO-01027 (dibenzazepine, DBZ), LY-411575, LY-4115
75, or LY2811376. In one embodiment, the high level of activin is greater than 40 ng/mL, greater than 50 ng/mL,
and levels above 75 ng/mL. In one embodiment, high levels of Activin are used during stage 3 or prior to the generation of pancreatic foregut endoderm cells. In one embodiment, low levels of Activin include levels below 30 ng/mL, below 20 ng/mL, below 10 ng/mL and below 5 ng/mL.
In one embodiment, low levels of activin are used during stage 4 or to generate PEC. In one embodiment, the endocrine gene that is inhibited or induced is NG
In another aspect, Activin A and Wnt3A are used alone or in combination to inhibit endocrine expression, preferably to inhibit NGN3 expression prior to generation of pancreatic foregut endoderm cells, or preferably during stage 3. In one aspect, a gamma sequestrantase inhibitor, preferably RO449, is used to induce expression of endocrine gene expression after generation of PEC, or preferably during stage 5, stage 6 and/or stage 7.
29097 or DAPT are used in the culture system.

An in vitro cell culture comprising endocrine cells, comprising at least 5% of said human cells.
% of the genes involved in insulin (INS), NK6 homeobox 1 (NKX6.1), pancreatic and duodenal homeobox 1 (PDX1), and transcriptome-associated loci (transcriptome-associated loci)
ion factor related locus 2 (NKX2.2), paired box 4 (PAX4), neurogenic differentiation
tion)1 (NEUROD), forkhead box A1 (FOXA1), forkhead box A2 (FOXA2), snail family zinc finger (snail
family zinc finger 2 (SNAIL2), and musculoaponeurotic fibrosarcoma oncogene family
The present invention relates to a method for the treatment of oncogene family (MAFA and MAFB) endocrine markers selected from the group consisting of neurogenin 3 (NGN3), islet 1 (ISL1), hepatocyte nuclear factor 6 (HNF6), GATA binding protein 4 (GATA binding protein 4), and the like.
ATA4), GATA binding protein 6 (GATA6), pancreatic specific transcription factor (pan
creas specific transcription factor) Ia (P
TF1A) and SRY (sex determining region Y)-9 (SOX9), and wherein said endocrine cells are unipotent and capable of maturing into pancreatic beta cells.

The endocrine cells are preferably immature beta cells.

It is preferred that at least 10% of the human cells are immature beta cells.

It is preferred that at least 20% of the human cells are immature beta cells.

It is preferred that at least 50% of the human cells are immature beta cells.

The cells are preferably non-recombinant.

The cells include human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), parthenogenetic cells, embryonic carcinoma cells (EC) cells, mesendoderm cells, definitive endoderm cells, PDX1-1 negative pancreatic foregut endoderm cells, PDX1 positive pancreatic foregut endoderm cells, and pancreatic endoderm cells (PECs).
Preferably, the cell is derived from a human pluripotent stem cell selected from the group consisting of:

1. A method for generating immature beta cells, comprising: a) contacting a population of PDX1-negative foregut endoderm cells with an agent that activates a TGFβ receptor family member, thereby generating PDX1-negative foregut endoderm cells that are capable of expressing a TGFβ receptor family member.
A method comprising the steps of: b) generating a PDX1-positive pancreatic endoderm cell population; and c) contacting the PDX-positive pancreatic endoderm cell population with a gamma-secretaase inhibitor, thereby generating immature beta cells that express INS and NKX6.1.

Preferably, a PDX1-negative foregut endoderm cell population is contacted with a Wnt family member and a heregulin family member.

TGF-beta receptor family members include activin A, activin B, and Nodal
, GDF-8, GDF-10, GDF-11 and GDF15.

TGF beta receptor family members include activin A, GDF-8 and GDF-
It is preferable that the number is 11.

The TGF beta receptor family member is preferably activin A.

Wnt family members are WNT1, WNT2, WNT2B, WNT3, and WNT3.
A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8
A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11,
It is preferably selected from the group consisting of WNT16 and WNT3A.

The Wnt family member is preferably Wnt3a.

Heregulin (HRG) family members are HRG1, HRG2, HRG3 and HR
It is preferably G4.

The HRG family members are preferably HRG1 and HRG2.

A method for producing insulin in vivo, comprising the steps of: a) contacting a PDX1-negative foregut endoderm cell population with an agent that activates a TGFβ receptor family member, thereby producing a PDX1-positive pancreatic endoderm cell population; and b) contacting the PDX1-positive pancreatic endoderm cell population with a gamma sequestrantase inhibitor, thereby producing insulin in vivo.
(c) generating immature beta cells that express X6.1; (b) implanting the immature beta cells from step (b); and (d) maturing the cells in vivo into mature beta cells that secrete insulin in response to glucose stimulation.

Preferred features and aspects of the present invention are as follows:

At stage 7, gene expression can still be regulated by agents. It is preferred to add a member of the FGF superfamily at stage 7.
It is preferable to add FGF2. FGF2 can increase SST expression and/or suppress INS expression, GCG expression or GHRL expression.
It is preferable to add MP. BMP suppresses INS expression, PDX1 expression, or IDI expression.
Expression can be increased.

Immature beta cell population that gives rise to in vivo function. The immature beta population is CHG
Preferably, the immature beta cell population is CHGA+. Preferably, the immature beta cell population is capable of functioning in vivo, i.e., secreting insulin in response to blood glucose when transplanted. Preferably, the endocrine cell population is capable of developing and maturing into functional pancreatic islet cells when transplanted. Preferably, the immature beta cell population is enriched for endocrine cells (or depleted of non-endocrine cells). In preferred embodiments, greater than about 50% of the cells in the immature beta cell population are CHGA+. In more preferred embodiments, greater than about 60%, or greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%, or greater than 98%, or 100% of the cells in the immature beta cell population are CHGA+. In preferred embodiments, less than about 50% of the cells in the immature beta cell population are CHGA-. In more preferred embodiments, less than about 15% of the cells in the immature beta cell population are CHGA-. In more preferred embodiments, less than about 10% or 50% of the cells in the immature beta cell population are CHGA-.
% or less than 3% or less than 2% or less than 1% or less than 0.5% or 0% is C
In addition, the expression of certain markers, such as NGN3 expression, is considered to be indicative of stage 3 HGA-.
During the course of the aging process, the generation of immature beta cells can be inhibited. Preferably, the immature beta cell population is monohormonal (e.g., INS only). The immature beta cell population is N
Preferably, the immature beta cell population is monohormonal and co-expresses other cell markers including NKX6.1 and PDX1. The immature beta cell population is monohormonal and preferably co-expresses other cell markers including NKX6.1 and PDX1. The immature beta cell population is monohormonal and preferably co-expresses other cell markers including NKX6.1 and PDX1.
It is preferred to have more single hormone expressing INS cells as a percentage of the population. It is preferred that the immature beta cell population has at least 50% single hormone expressing INS cells as a percentage of the total INS population. The immature beta cell population is CHGA+
In a preferred embodiment, more than about 25% of the cells in the immature beta cell population are CHGA+/INS+/NKX6.1+ (triple positive).
(triple positive). In a preferred embodiment, about 30% of the cells in the immature beta cell population
or more than 40% or more than 50% or more than 60% or more than 70% or more than 80% or more than 9
>0% or >95% or 100% are CHGA+/INS+/NKX6.1+ (triple positive).

The enriched immature beta cell population is made by dissociating and reaggregating the cell aggregates. Preferably, the enriched immature beta cell population is dissociated and reaggregated at stage 7. Preferably, the immature beta cell population is dissociated and reaggregated on about day 25 or day 26 or day 27 or day 28 or day 29 or day 30 or later.

Complex media is important for maintaining endocrine cells. Complex media is used by CMRL or RP
Preferably, the disease is MI.
It is preferable to use complex media at both stages 1 and 7. CMRL increases the expression of endocrine markers. RPMI increases the expression of endocrine markers.

Cryopreservation does not reduce the immature beta cell population at stage 7. Cryopreservation does not reduce CHGA+ expression at stage 7.

Stage 7 cells are NKX6.1 positive. Stage 7 cells are C-peptide positive. Stage 7 cells are NKX6.1 and C-peptide co-positive. Stage 7 cells are monohormonal. Stage 7 cells express INS and GCG alone.

Dissociation and reaggregation of stage 7 cells does not affect in vivo function.

Cryopreservation of stage 7 cells does not affect in vivo function.

Stage 7 cells can be cryopreserved and retain their in vivo function.

Increasing glucose concentration increases INS expression, GCG expression or SST expression.
Preferably, glucose concentrations are elevated at stage 6 or stage 7 or both. Preferably, GHRL expression is decreased as glucose concentrations are elevated.

In vitro immature beta cells that are monohormonal (INS only).

In vitro immature beta cells co-expressing NKX6.1 and PDX1.

Monohormonal (INS only) and co-expressing NKX6.1 and PDX1
In vitro immature beta cells.

In vitro immature beta cells that are monohormonal (INS only), co-express NKX6.1 and PDX1, and can mature in vivo into cells capable of secreting insulin in response to blood glucose.

In vitro immature beta cells that can mature in vivo into cells capable of secreting insulin in response to blood glucose.

In vitro immature beta cells that are CHGA+, INS+ and NKX6.1+. More than 10% of the total in vitro cell population is CHGA+, INS+ and NKX6.1
Preferably, the total in vitro cell population comprises immature beta cells that are +/- 50% of the total in vitro cell population.
Over 80% of the total in vitro cell population are immature beta, including immature beta cells that are CHGA+, INS+ and NKX6.1+. Over 80% of the total in vitro cell population are immature beta, including immature beta cells that are CHGA+, INS+ and NKX6.1+.

Purified immature beta cells. Purified immature beta cell precursors. Preferably, immature beta cells or beta cell precursors are purified from the stage 7 cell population using a zinc sensor. Preferably, faint fluorescence due to the presence of the Py1 sensor bound to zinc is enriched for beta cells. Preferably, bright fluorescence due to the presence of the Py1 sensor bound to zinc is enriched for alpha cells. Method for purifying beta cells using a zinc sensor. Method for purifying alpha cells using a zinc sensor. Purified alpha cells. Purified alpha cell precursors.

INS, IAPP, PDX1, NKX6.1, PAX4, PCSK1, G6PC2, G
CK, or enriched cell populations expressing markers of the beta cell lineage, such as SLC30A8.

Enriched cell populations expressing markers of the alpha cell lineage, such as GCG, ARX, or SLC30A8.

An enriched cell population in which more than 50% of the cells are INS positive. A cell population in which more than 90% of the cells are INS positive.
In a preferred embodiment, 50% to 99% of the cells are I positive.
These are NS positive cells.

A method for producing insulin in vivo, comprising: a) in vitro
b) obtaining a population of CHGA+ cells by transplanting the CHGA+ cells, thereby
and producing insulin in vivo.

It is preferable to dissociate and reaggregate CHGA+ cells at stage 7.

CHGA+ cells preferably express PDX1 and NKX6.1

CHGA+ cells preferably express INS and NKX6.1.

CHGA+ cells are preferably cultured in CMRL or RPMI.

CHGA+ cells are preferably cryopreserved and thawed prior to transplantation.

It is preferable to culture CHGA+ cells in a high glucose-containing medium.

Physiologically functional insulin-secreting cells can be generated in vivo
Vitro endocrine cells.

Physiologically functional insulin-secreting cells can be generated in vivo.
In vitro properly characterized immature beta cells.

Preferably, appropriately specified immature beta cells are dissociated at stage 7 and allowed to reaggregate.

Properly specified immature beta cells preferably express PDX1 and NKX6.1.

Properly specified immature beta cells preferably express INS and NKX6.1.

Appropriately specified immature beta cells are preferably cultured in CMRL or RPMI.

Preferably, appropriately identified immature beta cells are cryopreserved and thawed prior to transplantation.

Preferably, appropriately specified immature beta cells are cultured in high glucose containing medium.

A method for generating an enriched immature beta cell population in vitro, comprising the steps of obtaining and selecting a population of CHGA+ cells.A method for generating an enriched cell population in vitro that expresses INS, IAPP, PDX1, NKX6.1 or PAX4, comprising the steps of obtaining and selecting a population of CHGA+ cells.

A method for generating an enriched alpha cell population in vitro, comprising obtaining and selecting a population of CHGA+ cells.

A method for generating an enriched cell population expressing GCG or ARX in vitro, comprising obtaining and selecting a population of CHGA+ cells.

Preferably, enriched populations of alpha or beta cells are selected using a Py1 zinc sensor.

In some embodiments, the immature beta cells express INS and NKX6.1 and NGN.
In some embodiments, the immature beta cells do not substantially express INS, NKX6
.1 and MAFB, and does not substantially express NGN3.

1 is a photographic image of an aggregated suspension culture of dedifferentiated, reprogrammed cells, or cells also referred to herein as iPS cells. See Example 1.

2A-2C are bar graphs showing relative gene expression levels of OCT4 (FIG. 2A), BRACHYURY (FIG. 2B), CER1 (FIG. 2C), GSC (FIG. 2D), FOXA2 (FIG. 2E), FOXA1 (FIG. 2F), HNF6 (FIG. 2G), PDX1 (FIG. 2H), PTF1A (FIG. 2I), NKX6.1 (FIG. 2J), NGN3 (FIG. 2K), and INS (FIG. 2L). Expression levels are normalized to the mean expression levels of housekeeping genes, cyclophilin G, and TATA binding protein (TBP) expression. Graphs represent fold upregulation from the lowest data point in the data set. See Example 1.

Immunocytochemistry (ICC) micrographs of human iPS cell (E2021 using G4 hIPS cell line) cultures from stage 4 differentiation (PDX1 positive pancreatic endoderm cells) using antibodies specific for (Panel A) PDX-1, (Panel B) NKX6.1, (Panel C) PTF1A and (Panel D) Dapi. See Example 2.

Photographs of immunocytochemistry (ICC) of iPS cell cultures from stage 5 differentiation (E2021 using G4 hIPS cell line) using ligands specific for (Panel A) Glucagon, (Panel B) Insulin, (Panel C) Somatostatin and (Panel D) Dapi. See Example 2.

FIG. 5 shows an array location map and array key provided with the Proteome Profiler™ Human Phospho-RTK Antibody Array from R&D Systems. The location map in FIG. 5A shows the coordinates or locations of the RTK antibodies. The RTK family and the identity or name of the antibody are listed in the key in FIG. 5B. Thus, a positive signal observed in the developed film can be identified by overlaying a transparency as in FIG. 5A and identifying the signal by referencing the coordinates on the overlay (FIG. 5A) with the name of the RTK in FIG. 5B. See Example 4.

Figure 1 shows RTK array analysis of iPS cell-derived pancreatic endoderm cells (PEC) under four different conditions (Panels A, B, C and D as described in Example 5). Tyrosine phosphorylation of specific RTKs is observed by identification of high to low intensity signals. IGF 1R/IR and ERBB (EGFR) family members are identified or boxed. See Example 5.

Graphs showing human C-peptide and insulin concentrations in serum of transplanted mice from experiments E2314, E2356 and E2380 (FIG. 7A), E2347 (FIG. 7B) and E2354 (FIG. 7C). FIG. 7A: Treatment of PDX1-expressing cells with heregulin in vitro improves glucose-stimulated insulin secretion after transplantation and maturation in vivo. FIG. 7B: Glucose-stimulated C-peptide levels 23 weeks post-transplant, 3 weeks prior to STZ induction in a mouse diabetes model. Mice transplanted with PECs were analyzed for serum levels of human C-peptide at the indicated post-transplant time points, fasting, and 30 and 60 minutes after intraperitoneal glucose administration. In Figure 7C, PECs were encapsulated with a cell encapsulation device (Encaptra® EN20 or EN20, ViaCyte, San Diego, Calif.), and in some cases the device had microperforations (pEN20, ViaCyte, San Diego, Calif.). Such devices are described in U.S. Patent No. 8,278,106. See Example 7.

8A-8C are graphs showing the results of blood glucose analysis of STZ-treated mice from experiment #2347. Glucose-responsive insulin-secreting beta cells derived from transplanted iPECs control blood glucose in an STZ-induced diabetes model. FIG. 8A: Blood glucose for each of the 13 mice (baseline with and without Heregulin). FIG. 8B shows the combined average measurements for each treatment (baseline with and without Heregulin). Random non-fasting blood glucose level measurements are shown for 13 mice that received iPEC grafts up to 14 days prior to treatment with STZ (day 0), and for the same mice after STZ treatment and after explantation of the grafts. STZ-treated animals received STZ approximately 26 weeks after implantation of the graft (day 0). iPEC grafts were explanted (removed) 28 weeks after implantation of the graft, approximately 2 weeks after the start of STZ treatment. Non-fasting blood glucose measurements were collected over time for each animal. See Example 7.

9A-9E are bar graphs showing relative gene expression levels of PDX1 (FIG. 9A), NKX6.1 (FIG. 9B), PTF1A (FIG. 9C), NKX2.2 (FIG. 9D), and NGN3 (FIG. 9E). See Example 8.

13 is a photographic image of an aggregated suspension culture in which PDX1-negative foregut endoderm cells (stage 2) were differentiated into PDX1-positive foregut endoderm (stage 3) by the addition of 50 ng/mL activin A. See Example 8.

13A-13C are photographic images of aggregate suspension cultures at the end of stage 3 (day 8, top panel) and stage 4 (day 12, bottom panel) using 5 ng/mL (ACT5) or 10 ng/mL (ACT10) activin A at stage 3; or 5 ng/mL activin (ACT5) at stages 3 and 4. See Example 8.

Photographic images of aggregated suspension cultures during (A-E, top panels) or at the end of stage 4 (A-E, bottom panels). Top panels show that 10 ng/mL activin A and 2 ng/mL heregulin (ACT10 HGN2), or 20 ng/mL activin A and 2 ng/mL heregulin (ACT20 HGN2), or 20 ng/mL activin A and 10 ng/mL heregulin (ACT20 HGN10), or 10 ng/mL activin A, 2 ng/mL heregulin and 50 ng/mL WNT (ACT10 HGN2 WNT50) were added at stage 3. The bottom panel shows that 25 ng/mL activin A (ACT25), 50 ng/mL activin A (ACT50), 75 ng/mL activin A (ACT75) or 100 ng/mL activin A (ACT100) were added at stage 3. All conditions received low activin and heregulin at stage 4. See Example 9.

13A-C are bar graphs showing relative gene expression levels of NGN3 (FIG. 13A), NKX2.2 (FIG. 13B), and NKX6.1 (FIG. 13C) when activin, heregulin and WNT (AHW) were added at stage 3, and activin and heregulin (AH) were added at stage 4, as described in Example 9.

14A and 14B are bar graphs showing the relative gene expression levels of NKX6.1 (FIG. 14A) and NGN3 (FIG. 14B) when activin, heregulin and WNT (AHW) were added at stage 3, and activin and heregulin (AH) were added at stage 4, as described in Example 10.

1 is a graph showing the concentration of human C-peptide in serum of mice implanted with encapsulated PEC (control) and encapsulated modified PEC (generated with activin, heregulin and WNT (AHW) at stage 3 and activin and heregulin (AH) at stage 4). Expression levels were analyzed 11 weeks after implantation at fasting, 30 minutes, and 60 minutes after intraperitoneal glucose challenge. See Example 10.

1 is a graph showing the concentration of human C-peptide in serum of mice implanted with encapsulated PEC (control) and encapsulated modified PEC (generated with activin, heregulin and WNT (AHW) at stage 3 and activin and heregulin (AH) at stage 4). Expression levels were analyzed 14 weeks after implantation at fasting, 30 minutes, and 60 minutes after intraperitoneal glucose challenge. See Example 10.

1 is a bar graph showing relative gene expression levels of Neurogenin3 at days 13, 15, and 17 after treatment with a gamma secretase inhibitor at stage 5, as described in Example 11.

18A-D are bar graphs showing relative gene expression levels of PDX1 (FIG. 18A), NKX6.1 (FIG. 18B), SOX9 (FIG. 18C), and PTF1A (FIG. 18D) following differentiation according to Table 17 for stages 1-5, and for stages 6 and 7, only basal DMEM media plus FBS, matrigel, and rho kinase inhibitor were used as described in Example 12.

19A-D are bar graphs showing the relative gene expression levels of INS (FIG. 19A), GCG (FIG. 19B), GHRL (FIG. 19C) and SST (FIG. 19D) under the same differentiation conditions as described in Example 12 and as in FIG. 18.

1 is a graph showing the concentration of human C-peptide in the serum of mice implanted with encapsulated endocrine tissue prepared as described in Example 12 (E2395, Rag2, 10 week GSIS). Expression levels were analyzed 10 weeks after implantation, during fasting and 60 minutes after intraperitoneal glucose administration. See Example 12.

1 is a graph showing the concentration of human C-peptide in the serum of mice implanted with encapsulated endocrine tissue prepared as described in Example 12 (E2395, Rag2, 15 week GSIS). Expression levels were analyzed 15 weeks after implantation, during fasting and 60 minutes after intraperitoneal glucose administration. See Example 12.

22A-D are photomicrographs showing endocrine cell aggregates differentiated and matured in vivo as described in Example 12. Fields of cells are stained with DAPI (FIG. 22A) or antibodies against NKX6.1 (FIGS. 22B and 22C, nuclear staining) and chromogranin A (FIGS. 22B and 22D, cytoplasm). All images show the same field. See Example 12.

23A-C are photomicrographs showing endocrine cell aggregates as described in Example 12. Figure 23A shows a field of cells stained for INS (cytoplasm) and NKX6.1 (nucleus). Figure 23B shows the same field of cells stained for INS (cytoplasm) and PDX1 (nucleus). Figure 23C shows the same field of cells stained with DAPI. See Example 12.

24A-D are bar graphs showing Nanostring mRNA data of relative gene expression levels of INS (FIG. 24A), PDX1 (FIG. 24B), NKX6.1 (FIG. 24C) and ID1 (FIG. 24D) under differentiation conditions described in Example 13.

25A-C are photomicrographs showing endocrine cell aggregates as described in Example 13. Figure 25A is a control with no TT or BMP added, Figure 25B shows the addition of TT at stage 7, and Figure 25C shows the addition of BMP at stage 7. Figures 25A-C show staining for C-peptide (cytoplasmic) and PDX1 (nuclear).

26A and 26B are photomicrographs showing endocrine cell aggregates as described in Example 13. Figure 26A shows treatment with TT and BMP at stages 6 and 7, and Figure 26B shows addition of TT and BMP at stage 7. Figures 26A and 26B show staining for C-peptide (cytoplasmic) and PDX1 (nuclear).

27A-C show photomicrographs showing endocrine cell aggregates as described in Example 13, representing the same fields as in FIG. 25. FIG. 27A is a control with no TT or BMP added, FIG. 27B shows the addition of TT at stage 7, and FIG. 27C shows the addition of BMP at stage 7. FIG. 27A-C show staining for C-peptide (cytoplasmic) and NKX6.1 (nuclear).

28A-28B are photomicrographs showing endocrine cell aggregates as described in Example 13, representing the same fields as in FIG 26. FIG 28A shows treatment with TT and BMP at stages 6 and 7, and FIG 28B shows addition of TT and BMP at stage 7. FIG 28A and 28B show staining for C-peptide (cytoplasmic) and NKX6.1 (nuclear).

FIG. 13 is a bar graph showing relative gene expression levels of endocrine aggregates treated with nicotinamide (right bar; d21gi-d13-d15) or without nicotinamide treatment (left bar; d21gi-d13-d15+NICd15) at stage 6 as described in Example 13 and analyzed on day 21.

1 shows photographic images (days 21, 22, and 23) of endocrine cell aggregate suspension cultures that were dissociated and reaggregated beginning at stage 7 as described in Example 14, followed by addition of FBS alone, both FBS (panel A) and 0.05% Matrigel (MG; panel B), both FBS and 10 μM Y-27632 (Y; panel C), or all three: FBS, 0.05% Matrigel (MG), and 10 μM Y-27632 (Y; panel D).

31A-31D are bar graphs showing relative gene expression levels of NKX6.1 (FIG. 31A), NKX2.2 (FIG. 31B), PDX1 (FIG. 31C) and INS (FIG. 31D) comparing differentiation states with and without Noggin for days 1, 2 and 3. See Example 15.

32A-32C are bar graphs showing Nanostring mRNA data of relative gene expression levels of INS and GCG (FIG. 32A), GHRL and SST (FIG. 32B), and PDX1 and ID1 (FIG. 32C), depicting the effect of the indicated factors on gene expression. See Example 16.

33A-33C are bar graphs showing Nanostring mRNA data of relative gene expression levels for PDX1, NKX6.1, PTF1A, SOX9 (FIG. 33A); INS, GCG, PPY, GHRL (FIG. 33B), and PCSK1, GCK, SLC30A8, G6PC2 (FIG. 33C), describing endocrine (CHGA+) and non-endocrine (CHGA-) subpopulations in reaggregated cultures compared to non-reaggregated cultures. See Example 17.

FIG. 13 is a graph showing the concentration of human C-peptide in the serum of mice transplanted with encapsulated endocrine cells (re-agg sample) generated by dissociation and reaggregation at the onset of stage 7 enriched for endocrine (CHGA+) cell types as described in Example 18, compared to similarly differentiated but non-reaggregated cells (control sample). Expression levels were analyzed 21 weeks after transplantation, fasting, 30 minutes, and 60 minutes after intraperitoneal glucose challenge. See Example 18.

35A-C are bar graphs showing Nanostring mRNA data of relative gene expression levels of CHGA, INS, GCG, GHRL, PPY, SST (FIG. 35A); GCK, G6CP2, UCN3, PCSK1 (FIG. 35B); HNF1a, HNF4A, SLC30A8, CADH1 (FIG. 35C), illustrating the influence of DMEM and CMRL based media during stage 7. See Example 19.

36A and 36B are photomicrographs showing endocrine cell aggregates at stage 7 as described in Example 20. The aggregates have not reaggregated at stage 7. The same fields in Fig. 36A and Fig. 36B are stained for INS (cytoplasmic) and GCG (cytoplasmic), respectively; Fig. 36C is stained for NKX6.1 (nuclear) and C-peptide (cytoplasmic).

37A and 37B are photomicrographs showing endocrine cell aggregates at stage 7 as described in Example 20. The aggregates have reaggregated at stage 7. The same fields in Fig. 37A and Fig. 37B are stained for INS (cytoplasmic) and GCG (cytoplasmic), respectively; Fig. 37C is stained for NKX6.1 (nuclear) and C-peptide (cytoplasmic).

Graph showing human C-peptide concentration in serum of mice implanted with encapsulated endocrine cells produced as described in Example 20, where the cell cultures were frozen (cryopreserved) and thawed (F/T) at the beginning of stage 7 and not dissociated and reaggregated (left side), or they were not frozen (fresh), dissociated and reaggregated (right side). Expression levels were analyzed 12 weeks after implantation, fasting, 30 minutes and 60 minutes after intraperitoneal glucose challenge. See Example 21.

39A-B are bar graphs showing Nanostring mRNA data of relative gene expression levels of INS, GCG (FIG. 39A) and SST, GHRL (FIG. 39B) describing the effect of exogenous high glucose during stage 6 on endocrine hormone expression. See Example 21.

40A-40B are photomicrographs showing stage 7 endocrine cell aggregates with exogenous high glucose during stage 7 as described in Example 21. Figure 40A shows staining for INS (cytoplasmic) and Figure 40B shows staining for GCG (cytoplasmic).

FIG. 13 is a flow cytometry graph showing enrichment of immature endocrine beta cells from stage 7 by first treating with the zinc-binding agent Py1 and then sorting by fluorescence. See Example 24.

42A-C are bar graphs showing Nanostring mRNA data of relative gene expression levels of INS, GCG, GHRL, IAPP, SST and SST (FIG. 42A); PAX4, PDX1, ARX, NKX6.1 (FIG. 42B); and PCSK1, GCK, G6PC2 and SLC30A8 (FIG. 42C), characterizing and identifying cells enriched from zinc sorting. See Example 24.

Schematic diagram of stages 1-4 for the generation of pancreatic endoderm cells (PEC) in vitro and their development and maturation into insulin-secreting beta cells in vivo. The diagram also depicts the two main subpopulations of PEC, endocrine (CHGA+) and non-endocrine (CHGA-) cells. Embryonic stem cells (ESC), mesoendoderm (ME), definitive endoderm (DE), foregut (FG), posterior foregut (pFG), and pancreatic epithelium or pancreatic endoderm (PE).

Schematic diagram of stages 1-7 for the generation of endocrine cells in vitro. The diagram also depicts that high levels of Activin (stage 3) followed by low levels of Activin and removal of WNT (stage 4) suppresses or inhibits expression of NGN3, which is later expressed during stage 5 by addition of a gamma secretase inhibitor. Embryonic stem cells (ESC), mesoendoderm (ME), definitive endoderm (DE), foregut (FG), posterior foregut (pFG), and pancreatic epithelium or pancreatic endoderm (PE).

Schematic diagram of stages 1-7 for the generation of endocrine cells in vitro. The diagram also shows the stage, growth factors, cell type, and signature markers for each cell type: embryonic stem cell (ESC), mesoendoderm (ME), definitive endoderm (DE), foregut (FG), posterior foregut (pFG), pancreatic epithelium or pancreatic endoderm (PE), pre-beta cells (pre-B; or immature beta cells), and beta cells (β cells).

Detailed Description
The present invention can be easily understood by referring to the following detailed description of the preferred embodiment and examples of the present invention contained herein.However, before the disclosure and description of the present compounds, compositions and methods, it should be understood that the present invention is not limited to specific cell types, specific feeder cell layers, specific conditions, or specific methods, etc., and can vary as such.Many modifications and variations will be apparent to those skilled in the art.It should also be understood that the terminology used herein is merely for describing specific embodiments, and is not limiting.All patent and non-patent publication references are incorporated herein in their entirety by reference.

Various cell compositions derived from pluripotent stem cells are described herein and in Applicant's U.S. patent application Ser. No. 10/486,408, entitled METHODS FOR CULTURAL ENZYMES AND METHODS FOR TRANSFORMATION OF SUCCESSFUL HUMAN CARBOHYDRATES.
URE OF HESC ON FEEDER CELLS, filed August 6, 2002;
No. 11/021,618, entitled DEFINITIVE ENDODERM, 2004
No. 11/115,868, filed Dec. 23, 2003, entitled PDX1 EXPRESSION
G ENDODERM, filed April 26, 2005; Ser. No. 11/165,305, entitled METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIAL APPLICATIONS
ERENTIATING DEFINITIVE ENDODERM, June 2005
No. 11/573,662, filed on the 3rd, entitled METHODS FOR INCREAS
ING DEFINITIVE ENDODERM DIFFERENTIATION
OF PLURIPOTENT HUMAN EMBRYONIC STEM CELL
No. 12/729,084, entitled PDX1-EXPRESSING DORS, filed Aug. 15, 2005;
AL AND VENTRAL FOREGUT ENDODERM, filed October 27, 2005; Ser. No. 12/093,590, entitled MARKERS OF DEFINIT
IVE ENDODERM, filed on November 14, 2005; Ser. No. 11/993,399, entitled EMBRYONIC STEM CELL CULTURE COMPOSITIVE
ONS AND METHODS OF USE THEREOF, June 20, 2006
Application No. 11/588,693, entitled PDX1-EXPRESSING DOR
SAL AND VENTRAL FOREGUT ENDODERM, October 2006
Filed on the 27th of the month; No. 11/681,687, entitled ENDOCRINE PROGENI
TOR/PRECURSOR CELLS, PANCREATIC HORMONE-E
XPRESSING CELLS AND METHODS OF PRODUCTION
N, filed March 2, 2007; Ser. No. 11/807,223, entitled METHODS FOR
R CULTURE AND PRODUCTION OF SINGLE CELL
POPULATIONS OF HESC, filed May 24, 2007;
No. 3,944, entitled "METHODS OF PRODUCING PANCREATIC
HORMONES, filed July 5, 2007; 11/860,494, entitled METHODOLOGY
DS FOR INCREASING DEFINITIVE ENDODERM PR
ODUCTION, filed Sep. 24, 2007; Ser. No. 12/099,759, entitled ME
THOD OF PRODUCING PANCREATIC HORMONES, 2
No. 12/107,020, filed April 8, 2008, entitled METHODS FOR PARTICLES
URIFYING ENDORM AND PANCREATIC ENDORM
M CELLS DELIVERED FORM HUMAN EMBRYONIC STATE
M CELLS, filed April 21, 2008; Ser. No. 12/618,659, entitled ENC
APSULTATION OF PANCREATIC LINEAGE CELLS D
ERIVED FROM HUMAN PLURIPOTENT STEM CELLS
, filed Nov. 13, 2009;
No. 078, both with the title CELL COMPOSITIONS FROM DEDIFF
ERENTIATED REPROGRAMMED CELLS, filed April 22, 2010 and February 6, 2013;
ONS AND METHODS USEFUL FOR CULTURE DIF
FERENTIABLE CELLS, filed August 13, 2007;
No. 760, Title: STEM CELL AGGREGATE SUSPENSION CO.
MPOSITIONS AND METHODS OF DIFFERENTIATION
N THEREOF, filed November 4, 2008; Serial No. 13/259,15, entitled SM
ALL MOLECULES SUPPORTING PLURIPOTENT CEL
L GROWTH, filed April 27, 2010; PCT/US11/25628, entitled L
Loading System for an Encapsulation Device
E, filed February 21, 2011; Ser. No. 13/992,931, entitled AGENTS AN
D METHODS FOR INHIBITING PLURIPOTENT STATE
M CELLS, filed December 28, 2010; and U.S. Design Application No. 29/408,3
No. 29/408,368, filed December 12, 2011; No. 29/423,365, filed May 31, 2012; and No. 29/426,365, filed May 31, 2012.
/447,944, filed March 13, 2013; and U.S. Provisional Patent Application No. 61/774
, No. 443, entitled "SEMIPERMEABLE MACRO IMPLANTABLE"
CELLULAR ENCAPSULATION DEVICES, November 7, 2013
Application No. 61/775,480, entitled CRYOPRESERVATION, HIB
ERATION AND ROOM TEMPERATURE STORAGE OF
ENCAPSULATED PANCREATIC ENDODERM CELL A
GGREGATES, filed March 8, 2013; and 61/781,005, entitled IN
VITRO DIFFERENTIATION OF PLURIPOTENT ST
EM CELLS TO PANCREATIC ENDODERM CELLS (PE
C) AND ENDOCRINE CELLS, filed March 14, 2013.

DEFINITIONS Numerical ranges expressed herein will be understood to be inclusive of the stated endpoints and to recite every integer between the endpoints of the stated numerical range.

Unless otherwise specified, the terms used herein are understood according to conventional usage by those skilled in the art.Also, for the purposes of this specification and the appended claims, unless otherwise specified, all amounts of ingredients, percentages or proportions of materials, numbers expressing reaction conditions, and other numerical values used in this specification and the appended claims should be understood to be modified in all cases by the term "about".Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that can vary depending on the desired properties to be obtained by the present invention.
At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of significant digits given and by applying ordinary rounding techniques.

The practice of the embodiments described herein will employ, unless otherwise specified, conventional techniques of cell biology, molecular biology, genetics, chemistry, microbiology, recombinant DNA, and immunology.

The term "cell" as used herein refers to an individual cell, a cell line, or a culture derived from such a cell. A "culture" refers to a composition containing isolated cells of the same or different types. A "culture", "population" or "cell population" as used herein can be and are used interchangeably, with the meaning being clear depending on the context. For example, the term "population" can refer to a cell culture of two or more cells with the same distinguishing characteristic, or a culture of two or more cell types with different distinguishing characteristics, e.g., a population in one context can be a subpopulation in another context. The term "subpopulation", when used to describe a particular cell type in a cell culture or cell population, refers to a subset of the cell culture or cell population.

As used herein, the phrase "totipotent stem cells" refers to cells that have the ability to differentiate into all cells that make up an organism, such as cells resulting from the fusion of an egg cell and a sperm cell. Cells resulting from the first few divisions of a fertilized egg may also be totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells, such as ES cells, can give rise to any fetal or adult cell type. However, they cannot develop into a fetus or an adult animal on their own, as they lack the potential to develop into extraembryonic tissues. Extraembryonic tissues are derived, in part, from the extraembryonic endoderm and are called distal endoderm.
endoderm (Reichert's membrane) and visceral endoderm
The distal and proximal endoderm (which forms part of the yolk sac) can be further classified into the distal endoderm (which forms part of the yolk sac) and the proximal endoderm (which forms part of the yolk sac). Both the distal and proximal endoderm support the development of the embryo, but do not themselves form embryonic structures. Other extraembryonic tissues also exist, including the extraembryonic mesoderm and extraembryonic ectoderm.

In some embodiments, "pluripotent cells" are used as starting material for differentiation into endoderm lineages, and more particularly, pancreatic endoderm-type cells. As used herein, "pluripotent" refers to
Or "pluripotent cells" or equivalents refer to cells that can grow in cell culture and differentiate into various lineage-restricted cell populations that exhibit pluripotent properties, for example, both pluripotent ES cells and induced pluripotent stem (iPS) cells can give rise to each of the three embryonic cell lineages. However, pluripotent cells cannot generate an entire organism, i.e., pluripotent cells are not totipotent.

In certain embodiments, the pluripotent cells used as starting material are hES cells, hE
As used herein, "embryonic" refers to a region of developmental stages of an organism that begins with a single zygote and ends with a multicellular structure that no longer contains pluripotent or totipotent cells, except for the gamete cells that have developed. The term "embryonic" refers to embryos derived by gamete fusion as well as embryos derived by somatic cell nuclear transfer. In yet another embodiment, the pluripotent cells are not derived or directly derived from an embryo, e.g., iPS cells are derived from non-pluripotent cells, e.g., multipotent cells or terminally differentiated cells. Human pluripotent stem cell lines as used herein include hESCs and iPSCs, e.g., CyT49, CyT25, CyT203, CyT212, BG01, CyT203, CyT212 ...
, BG02, BG03, or any of those listed in Tables 4 and 5, or any of those now known or publicly available or later made.

Human pluripotent stem cells may also be defined or characterized by the presence of several transcription factors and cell surface proteins, including the transcription factors Oct-4, Nanog, and Sox-2, which form a core regulatory complex that ensures the repression of genes that lead to differentiation and the maintenance of pluripotency, and cell surface antigens such as the glycolipids SSEA3, SSEA4 and the keratin sulfate antigens, Tra-1-60 and Tra-1-81, and alkaline phosphatase.

As used herein, "induced pluripotent stem cells" or "iPS cells" or "iPS cells"
The phrase "SC" refers to non-pluripotent cells, generally adult somatic cells, or, for example, fibroblasts,
It refers to a type of pluripotent stem cell artificially prepared by inserting a specific gene or gene product called a reprogramming factor from a terminally differentiated cell such as a hematopoietic cell, a muscle cell, a neuron, or an epidermal cell. Takahashi et al., Cell, 131:861-
872 (2007); Wernig et al., Nature, 448:318-324 (200
7); Park et al., Nature, 451:141-146 (2008). These and subsequently published methods for generating iPSCs are well known and include the use of iPSCs in a wide variety of applications.
How the PSCs are derived or generated is not a limitation of the invention. Induced pluripotent stem cells are characterized by the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and developmental and differentiation potential (diff) from naturally occurring human pluripotent stem cells, such as hES cells.
Human iPS cells are substantially similar in many respects, including the ability to differentiate into multiple organs. Human iPS cells provide a source of pluripotent stem cells without the use of embryos.

As used herein, "reprogramming" means
"Reprogrammed" or its equivalent means that the ability of a cell to form progeny of at least one new cell type is lost in culture or in vivo.
pluripotent cell reprogramming refers to a process in which the cell's pluripotency is measurably increased over what it would have under the same conditions without reprogramming. In certain embodiments described herein, somatic cells are "reprogrammed" into pluripotent cells. In certain aspects, somatic cells are reprogrammed when, after sufficient proliferation, a measurable proportion of cells exhibit phenotypic characteristics of the new pluripotent cell type, either in vivo or in vitro cell culture. Without reprogramming, such somatic cells would not give rise to progeny that exhibit phenotypic characteristics of the new pluripotent cell type. If somatic cells can give rise to progeny that exhibit phenotypic characteristics of the new pluripotent cell type without reprogramming, the proportion of progeny derived from these somatic cells that exhibit phenotypic characteristics of the new pluripotent cell type will be measurably increased over that before reprogramming.

As used herein, the phrase "differentiation programming" refers to a process in which cells are changed to form at least one new cell type progeny with a new differentiation state, either in culture or in vivo, compared to the same condition without differentiation reprogramming. This process includes differentiation, dedifferentiation and transdifferentiation. Thus, as used herein, the phrase "differentiation" refers to the process in which a less specialized cell becomes a more specialized cell type. In contrast, the phrase "dedifferentiation" refers to the process in which a partially or terminally differentiated cell reverts to an earlier developmental stage, such as a pluripotent or multipotent cell. In further contrast, the phrase "transdifferentiation" refers to the process in which one differentiated cell type is transformed into another differentiated cell type.

As used herein, "multipotency" or "multipotent cells" or equivalents refer to a cell type that can give rise to a limited number of other specific cell types, i.e., multipotent cells are committed to one or more embryonic cell fates and thus, in contrast to pluripotent cells, are unable to give rise to each of the three embryonic cell lineages, as well as extraembryonic cells.
Multipotent somatic cells are more differentiated than pluripotent cells, but are not terminally differentiated. Thus, pluripotent cells have a higher developmental potential than multipotent cells. Developmental potential determining factors that can be used to reprogram somatic cells or generate iPS cells include, but are not limited to, Oct-4, Sox2, FoxD3, UTF1, Stella
, Rexl, ZNF206, Soxl5, Mybl2, Lin28, Nanog, DPP
A2, ESG1, Otx2 or a combination thereof.

As used herein, "unipotent" or "unipotent
"Unipotent nipotency" or "unipotent cells" or equivalents refer to a cell type that can give rise to only one other lineage cell. For example, immature beta cells as described herein have the capacity to differentiate only into insulin beta cells and do not have the potential to differentiate into, for example, glucagon (alpha) cells, somatostatin (delta) cells and pancreatic polypeptide (gamma) cells. In contrast, endocrine progenitor cells, PDX1-positive pancreatic endoderm cells, definitive endoderm cells, or mesendoderm cells, and hES cells are all multipotent or pluripotent (hES) cells that can give rise to pancreatic alpha, beta, delta and gamma islet cells, respectively.
C) cells. Similarly, pancreatic alpha, beta, delta and gamma islet cells are endocrine precursor cells, PDX1-positive pancreatic endoderm cells, definitive endoderm cells, or mesendoderm cells, and lineage cells of hES cells.

As used herein, the terms "monohormonal" and "polyhormonal" refer to
"ERBB receptor tyrosine kinase activating agents" as used herein include, but are not limited to, those that express at least 16 different EGF family ligands that bind to the ERBB receptor: EGF family ligands, including but not limited to those that express only one hormone (e.g., immature beta cells and beta cells, which express only insulin protein and no glucagon or somatostatin protein), or cells that express more than one or multiple hormones (e.g., endocrine precursor or progenitor cells having subpopulations of cells that express two, three, or four or more hormones on the same cell).
GF (epidermal growth factor), AG or AREG (amphiregulin), and TGF-alpha (transforming growth factor-alpha), Btc (betacellulin), HBEGF (heparin-binding EGF, and Ereg (epiregulin)), neuregulin-1, -2
Neughrelin (or heregulin), such as heregulin-1, -2, -3 and -4 (or heregulin-1, -2, -3 and -4). However, in the present invention, any one of the four ERBB receptors is included.
Contemplated are any ligands capable of binding to one or a combination thereof to induce the formation of homo- and heterodimeric receptor complexes that lead to activation and subsequent phosphorylation of the intrinsic kinase domain. See also Table 13.

The term "cell lineage," as used herein, refers to all stages of development of a cell type, from the earliest progenitor cells to fully mature (i.e., specialized) cells. For example, "definitive endoderm-lineage cells" or "PDX1-negative endoderm-lineage cells" or "PDX1-negative endoderm-lineage cells" are used to refer to the development of a cell type.
"PDX1-positive pancreatic endoderm lineage cells" or "endocrine precursor lineage cells" or "endocrine lineage cells" or "immature beta lineage cells" and the like refer to cells derived or differentiated from definitive endoderm cells, PDX1-negative endoderm cells, PDX1-positive pancreatic endoderm cells, and the like. Definitive endoderm cells are lineages of mesendoderm cells, one of whose precursors. PDX-1-positive pancreatic endoderm cells are lineages of definitive endoderm cells, one of whose precursors. PDX1
Endocrine precursors within the lineages of PDX1 positive pancreatic cells, definitive endoderm cells, and mesendoderm cells are all precursors thereof. Endocrine precursors, PDX1 positive pancreatic cells, immature beta cells within the lineages of definitive endoderm cells, and mesendoderm cells are all precursors thereof. Beta cells, for example, are the only lineage of immature beta cells. Furthermore, all of the endoderm lineage cells described herein are derived from hES cells.
It is a lineage cell.

"Precursor cells" or "progenitor cells", as used herein, may generally be any cell in a cell differentiation pathway that can differentiate into a more mature cell. As such, precursor cells may be pluripotent cells, partially differentiated multipotent cells, or reversibly differentiated cells. The term "precursor cell population" refers to a group of cells that can develop into a more mature or differentiated cell type. Precursor cell populations may include pluripotent cells, cells restricted to stem cell lineages (i.e., cells that cannot develop into all of the ectodermal lineages, or cells that can only develop into cells of, for example, neuronal lineages), and cells that are reversibly restricted to stem cell lineages. Thus, the term "precursor cells" or "progenitor cells" may be "pluripotent cells" or "multipotent cells".

"Endocrine precursor/progenitor cells" as used herein refer to cells that contain at least one of the following: neurogenin 3 (NEUROG3), PDX1, PTF1A, SOX9, NKX6.1, HNF1
b, GATA4, HNF6, FOXA1, FOXA2, GATA6, MYT1, ISLE
T1, NEUROD, SNAIL2, MNX1, IA1, RFX6, PAX4, PAX6
Endocrine precursor/progenitor cells refer to multipotent cells of the definitive endoderm lineage that express markers from the list consisting of: NKX2.2, MAFA and MAFB, which can be further differentiated into cells of the endocrine lineage, including but not limited to pancreatic islet hormone expressing cells.
They are unable to differentiate into as many different cell, tissue and/or organ types as compared to less specifically differentiated definitive endoderm lineage cells, such as PDX1 positive pancreatic endoderm cells or definitive endoderm cells or mesendoderm cells. Endocrine precursor/progenitor cells are described in detail at least in Applicant's U.S. Patent No. 8,129,182.

As used herein, the terms "develop from pluripotent cells", "differentiate from pluripotent cells", "mature from pluripotent cells" or "generate from pluripotent cells", "derived from pluripotent cells", "differentiate from pluripotent cells" and equivalent expressions are intended to be understood as meaning the same as, for example, those described in International Patent Application No. PCT/US2007/015536, entitled METHODS OF PRACTICE
In the case of endocrine cells maturing from transplanted PDX1 pancreatic endoderm cells in vivo, as described in ODUCING PANCREATIC HORMONES,
"Pluripotent" refers to the generation of differentiated cell types from pluripotent cells in vitro or in vivo. All such terms refer to the progression of a cell from a stage with the potential to differentiate into at least two different cell lineages to becoming a specialized and terminally differentiated cell. Such terms may be used interchangeably for the purposes of this application. In the embodiments described herein, such differentiation is made reversible, thus allowing the generation of pluripotent or at least two distinct cell lineages.
Culture conditions are contemplated that allow for selective reversion of the capacity to differentiate into one or more cell lineages.

The term "feeder cells" refers to cell cultures that grow in vitro, secrete at least one factor into the cell culture, and can be used to support the growth of other cells of interest in the culture. As used herein, "feeder cell layer" can be used interchangeably with the term "feeder cells." Feeder cells may comprise a monolayer that covers the surface of a culture dish in one complete layer before they begin to grow on top of each other, or may comprise clusters of cells. In a preferred embodiment, the feeder cells comprise an adherent monolayer.

Similarly, embodiments in which pluripotent cell cultures or pluripotent aggregate suspension cultures are grown in defined conditions or culture systems without the use of feeder cells are "feeder-free." Feeder-free culture methods increase the scalability and reproducibility of pluripotent cell cultures and reduce the risk of contamination with infectious agents, for example, from feeder cells or other animal-sourced culture components. Feeder-free methods are also known as B
U.S. Pat. No. 6,800,480 to Odnar et al.
However, in contrast to the '480 patent, the embodiments described herein, whether pluripotent, multipotent or differentiated cell cultures, are feeder-free and do not further contain endogenous or exogenous extracellular matrix; that is, the cultures described herein are not only feeder-free, but also extracellular matrix-free. See, e.g., U.S. Pat. No. 6,800,480.
In U.S. Pat. No. 6,800,480, extracellular matrix is prepared by culturing fibroblasts, lysing the fibroblasts in situ, and then washing what remains after lysis. Alternatively, U.S. Pat. No. 6,800,480 describes collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin sulfate, chondroitin sulfate, dermatan sulfate,
The extracellular matrix may also be prepared from isolated matrix components or combinations of components selected from aggrecan, biglycan, thrombospondin, vitronectin, and decorin. In the embodiments described herein, the extracellular matrix is not generated by growing a feeder layer or fibroblast layer and lysing the cells to generate the extracellular matrix, but rather by first selecting collagen, placental matrix, fibronectin,
There is also no need to coat tissue culture vessels with an extracellular matrix component or combination of extracellular matrix components selected from laminin, merosin, tenascin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin. Thus, the aggregate suspension cultures described herein for pluripotent, multipotent, and differentiated cells do not require a feeder layer, lysed feeder cells, or fibroblasts that generate an extracellular matrix coating, exogenously added extracellular matrix or matrix components, and are described in International Patent Application PubMed.
CT/US2008/080516, entitled METHODS AND COMPOSITIONS
ONS FOR FEEDER-FREE PLURIPOTENT STEM CEL
As described in L MEDIA CONTAINING HUMAN SERUM:
The use of soluble human serum components overcomes the need for feeder cells or feeder monolayers, as well as the need for endogenous extracellular matrix derived from feeder cells or fibroblasts or exogenously added extracellular matrix components.

As used herein, the terms "cluster" and "clump" or "aggregate" can be used interchangeably and generally refer to a group of cells that have not aggregated to form clusters or have close cell-cell contacts after dissociation into single cells. The term "reaggregation" as used herein refers to dissociating clusters, clumps and/or aggregates into smaller clusters, clumps and/or aggregates or single cells and then reaggregating the clusters, clumps and/or aggregates to form new cell-cell contacts. This disaggregation is typically manual in nature (e.g., using a Pasteur pipette), although other means of disaggregation are contemplated. Aggregated suspension pluripotent or multipotent cell cultures are substantially similar to those described in WO 2005/023666.
T/US2007/062755, entitled COMPOSITIONS AND METHODS
DS FOR CULTURE DIFFERENTIAL CELLS" and P
CT/US2008/082356, entitled "STEM CELL AGGREGATE
SUSPENSION COMPOSITIONS AND METHODS OF D
As described in IFFE RENTIATION THEREOF.

The term "single cell suspension" or its equivalent refers to a pluripotent, multipotent or terminally differentiated single cell suspension, or a single cell suspension derived from pluripotent or multipotent cells by any mechanical or chemical means. There are several methods for dissociating cell clusters from primary tissues, cells in attached cultures, and aggregates to form single cell suspensions, such as physical forces (mechanical dissociation, such as cell scrapers, trituration through narrow-bore pipettes, fine needle aspiration, vortex disaggregation and forced filtration through fine nylon or stainless steel mesh), enzymes (enzymatic dissociation, such as trypsin, collagenase, Accutase™), or a combination of both. Furthermore, methods and culture medium conditions that can support the dissociation of pluripotent, multipotent or differentiated cells into single cells are useful for expansion, cell sorting, and defined seeding for multi-well plate assays, and also allow automation of culture procedures and clonal expansion.

In a preferred embodiment, the culture method or culture system does not include any animal-sourced products. In another preferred embodiment, the culture method is xeno-free. In a further preferred embodiment, the culture method described herein meets or exceeds one or more conditions or requirements for commercial manufacturing of human cell therapeutics.

The population of pluripotent cells can be further cultured in the presence of certain supplemental growth factors to obtain a population of cells that are or will develop into different cell lineages, or can be selectively reversed to be capable of developing into different cell lineages. The term "supplemental growth factor" is used in its broadest context and refers to a substance that is effective to promote the growth of pluripotent cells, to maintain cell survival, to stimulate cell differentiation, and/or to stimulate the reversal of cell differentiation. Furthermore, supplemental growth factors can be substances secreted from feeder cells into the medium. Such substances include, but are not limited to, cytokines, chemokines, small molecules, neutralizing antibodies, and proteins. Growth factors can also include intercellular signaling polypeptides that control the development and maintenance of cells and the morphology and function of tissues. In a preferred embodiment, the supplemental growth factors are selected from the group consisting of steel cell factor (SCF), oncostatin M (
OSM), ciliary neurotrophic factor (CNTF), interleukin-6 (IL-6) in combination with soluble interleukin-6 receptor (IL-6R), fibroblast growth factor (FGF)
, bone morphogenetic proteins (BMPs), tumor necrosis factors (TNFs), and granulocyte-macrophage colony stimulating factors (GM-CSFs).

In certain processes for generating the cells described herein, growth factors are removed from the cell culture or cell population after addition. For example, activin A, activin B, GD
Growth factors such as activin, F-8, or GDF-11 can be added and removed within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days after their addition. In some embodiments, differentiation factors are not removed from the cell culture per se, but are removed by omitting them from the cell culture medium, e.g., changing cell culture medium that contained activin to medium that does not contain activin.

The efficiency of the differentiation process can be adjusted by modifying certain parameters, including, but not limited to, cell growth conditions, growth factor concentrations, and timing of culture steps, so that the differentiation procedures described herein can result in a differentiation efficiency of about 5%, about 10%, about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%, or about 150%, or about 250%, or about 300%, or about 400%, or about 500%, or about 600%, or about 1000%, or about 1500%, or about 2500, or about 3000, or about 4000, or about 5000, or about 6000, or about 10000, or about 15000, or about 25000
%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55
%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95
%, or about 95% or more of pluripotent cells, including induced pluripotent cells, can be converted to multipotent or differentiated cells, such as definitive endoderm. Furthermore, the conversion rate or efficiency can be measured by comparing the rate of differentiation of one type of multipotent cell to a further differentiated multipotent cell, e.g.
Definitive endoderm to foregut endoderm, PDX1-positive foregut endoderm, PDX1-positive pancreatic endoderm or P
DX1/NKX6.1 co-positive pancreatic endoderm, endocrine precursors/precursors or NGN3/NK
X2.2 co-positive endocrine precursors/precursors, and hormone-secreting endocrine cells or INS;
It may also refer to differentiation into GCG, GHRL, SST, PP single positive endocrine cells. In processes using isolation of preprimitive streak or mesendoderm cells, a substantially pure population of preprimitive streak or mesendoderm cells can be recovered.

General methods for generating endoderm-lineage cells derived from hES cells are described in the related US patent applications mentioned above, as well as in D'Amour et al., 2005 Nat Biotechnol.
ol. 23:1534-41, published online October 28, 2005; D'Amour
et al., 2006 Nat Biotechnol. 24(11):1392-401, 20
Published online October 19, 2006; Kroon et al. (2008) Nat Biotech
No. 26(4):443-452, published online February 20, 2008; Kell
(2011) Nat. Biotechnol. 29(8):750-6, published online July 31, 2011; and Schulz et al. (2012) PLosOne, 7(5
): e37004, published online on May 18, 2012.
described a five-stage differentiation protocol: stage 1 (resulting in the production of mostly definitive endoderm), stage 2 (resulting in the production of mostly PDX1-negative foregut endoderm), stage 3 (resulting in the production of mostly PDX1-positive foregut endoderm), stage 4 (resulting in the production of mostly pancreatic endoderm or pancreatic endocrine precursors) and stage 5 (resulting in the production of mostly hormone-expressing endocrine cells).

The term "trophectoderm" refers to HAND1, Eomes, MASH2, ESXL1, H
CG, KRT18, PSG3, SFXN5, DLX3, PSX1, ETS2, and ER
BB gene expression in non-trophectoderm cells or cell populations, HAND1, Eomes, MASH2, ESXL1, HCG, KRT18, PS
G3, SFXN5, DLX3, PSX1, ETS2, and ERBB expression levels.

The term "extraembryonic endoderm" refers to a cell or population of cells that is differentiated from the extraembryonic endoderm by the expression level of a marker selected from the group consisting of SOX7, SOX17, THBD, SPARC, DAB1, or AFP genes.
, THBD, SPARC, DAB1, or AFP.

The term "preprimitive streak cells" refers to cells in which the expression levels of the FGF8 marker gene and/or the NODAL marker gene are BRACHURY low, FGF4 low, SNAI1 low,
low, SOX17 low, FOXA2 low, SOX7 low and SOX1
It refers to relatively high pluripotency cells compared to low.

The term "mesendoderm cells" refers to the cells that express the brachyury marker gene, FGF4, SN
The expression levels of the AI1, MIXL1 and/or WNT3 marker genes are
low, CXCR4 low, FOXA2 low, SOX7 low and SOX1
It refers to relatively high pluripotency cells compared to low.

The term "definitive endoderm (DE)" refers to multipotent endoderm lineage cells that can differentiate into cells of the gut or gut-derived organs. According to certain embodiments, the definitive endoderm cells are mammalian cells, and in preferred embodiments, the definitive endoderm cells are human cells. In some embodiments of the invention, the definitive endoderm cells express certain markers or fail to significantly express certain markers. In some embodiments, the S
OX17, CXCR4, MIXL1, GATA4, HNF3β, GSC, FGF17, V
One or more markers selected from WF, CALCR, FOXQ1, CMKOR1 and CRIP1 are expressed in definitive endoderm cells.
, alpha-fetoprotein (AFP), thrombomodulin (TM), SPARC,
One or more markers selected from SOX7 and HNF4alpha are not expressed or are not significantly expressed in definitive endoderm cells. Definitive endoderm cell populations and methods for producing same are described in U.S. Patent Application Serial No. 11/021,618, entitled DEFINITIV
E ENDODERM, filed December 23, 2004.

The term "PDX1-negative foregut endoderm cells" or "foregut endoderm cells" or equivalents thereof refer to the SOX17 marker, HNF1β (HNF1B) marker, HNF4 alpha (HNF4α) marker,
NF4A) and FOXA1 markers, but not PDX1, AFP, or SOX
PDX1-negative foregut endoderm cell populations and methods for producing the same are described in U.S. patent application Ser. No. 11/588,693, entitled PDX1-
expressing dorsal and ventral foregut en
doderm, filed October 27, 2006.

"PDX1 positive, dorsally biased foregut endoderm cells" (dorsal PDX1 positive foregut endoderm cells)
Alternatively, the term "PDX1 positive endoderm" or its equivalents may be used in conjunction with U.S. patent application Ser. No. 11/588,693, entitled PDX1 EXPRESSING DOSAL A, and/or its equivalents.
No. 11/115,868, entitled PDX1-expression ND VENTRAL FOREGUT ENDODERM, filed on October 27, 2006, and U.S. Patent Application No.
The cells express one or more markers selected from Table 1 and/or one or more markers selected from Table 2, which are also described in US Pat. No. 6,399,323, filed Apr. 26, 2005, filed by Gen Endoderm.

"Pancreatic endoderm", "pancreatic epithelium",
The terms "pancreatic epithelium" (which may all be abbreviated as "PE"), "pancreatic progenitors", "PDX-1 positive pancreatic endoderm" or equivalents thereof such as "pancreatic endoderm cells" (PECs) are all precursor or progenitor pancreatic cells. As described herein, PECs are a population of progenitor cells following stage 4 differentiation (about days 12-14) and are divided into at least two major distinct populations: i) they express PDX1 and NKX6.1, but
and ii) polyhormonal endocrine cells (CHGA positive, CHGA+), or "endocrine multipotent progenitor subpopulation (CHGA+)," or "endocrine (CHGA+) subpopulation" or "endocrine (CHGA+) cells" or equivalents. PEC pancreatic progenitor subpopulations that express PDX1 and NKX6.1, but do not express CHGA, are also referred to as "non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-)" or "non-endocrine progenitor subpopulation,""non-endocrine (CHGA-) subpopulation,""non-endocrine (CHGA-) subpopulation,""multipotent progenitor subpopulation," and the like. The PEC polyhormonal endocrine cell subpopulation that expresses CHGA is also referred to as "endocrine lineage committed cells (CHGA+) or endocrine cells" or "CHGA+ cells". Without being bound by theory, it is hypothesized that the cell population that expresses NKX6.1 but not CHGA is the more active and therapeutic PEC component, while the population of CHGA positive polyhormonal endocrine cells is hypothesized to further differentiate and mature into glucagon expressing islet cells in vivo. Kelly et al. (2011) Cell-surface marrow swarm analysis of endocrine cells.
kers for the isolation of panicked cell
l types derived from human embryonic stem
m cells, Nat Biotechnol. 29(8):750-756, 201
Published online July 31, 2011 and Schulz et al. (2012), A Scalable
e System for Production of Functional Pa
Creative Progenitors from Human Embryony
c See Stem Cells, PLosOne 7(5):1-17, e37004.

Furthermore, pancreatic endoderm cells are sometimes used to refer not to PECs as described immediately above, but generally to at least stage 3 and stage 4 type cells. This usage and meaning will be clear from the context. Thus, pancreatic endoderm derived from pluripotent stem cells, and at least hES and hIPS cells, is distinct from other endodermal lineage cell types, as are PD
The expression of markers selected from the group consisting of markers X1, NKX6.1, PTF1A, CPA1, cMYC, NGN3, PAX4, ARX and NKX2.2 is differentiated based on differential or high levels of expression, while genes characteristic of pancreatic endocrine cells, such as CHGA, INS, G
CG, GHRL, SST, MAFA, PCSK1 and GLUT1 are not substantially expressed. Furthermore, some "endocrine precursor cells" that express NGN3 can differentiate into other non-pancreatic structures (e.g., duodenum). Pancreatic endoderm or endocrine precursor cell populations and methods thereof are described in U.S. Patent Application Serial No. 11/773,944, entitled Methods of Producing Endocrine Progenitor Cells.
No. 12/107,020, entitled METHODS FOR PURIFICATION
ING ENDODERM AND PANCREATIC ENDODERM CEL
LS DELIVERED FORM HUMAN EMBRYONIC STEM CEL
LS, filed April 21, 2008.

"Endocrine cells" or "pancreatic islet hormone expressing cells", "pancreatic endocrine cells", "pancreatic islet cells"
The term "pancreatic islet" or equivalents refer to cells that may be polyhormonal or monohormonal. Thus, the cells can express one or more pancreatic hormones that have at least some of the functions of human pancreatic islet cells. Pancreatic islet hormone-expressing cells may be mature or immature and are more differentiated or developmentally committed than the endocrine precursor/progenitor type cells from which they are derived.

As used herein, the phrase "immature endocrine cells," including "stage 7 cultures" or "immature beta cells," refers to cells that are in
Refers to an in vitro generated endocrine cell population that can function in vivo, e.g., immature beta cells that secrete insulin in response to blood glucose when transplanted. The properly specified endocrine cells or stage 7 cultures may have additional properties including: when transplanted, the properly specified endocrine cells can develop and mature into functional pancreatic islet cells. The properly specified endocrine cells may be enriched for endocrine cells (or depleted of non-endocrine cells). In a preferred embodiment, greater than about 50% of the cells in the properly specified endocrine cell population are CHGA+. In a more preferred embodiment, greater than about 60%, or greater than 70%, or greater than 80% of the cells in the properly specified endocrine cell population are CHGA+.
In a preferred embodiment, less than about 50% of the cells in a properly specified endocrine cell population are CHGA+.
In a more preferred embodiment, less than about 15% of the cells in a properly specified endocrine cell population are CHGA-. In a more preferred embodiment, less than about 10%, or less than 5%, or less than 3%, or less than 2%, or less than 1% of the cells in a properly specified endocrine cell population are CHGA-.
Less than or 0.5% or 0% are CHGA-. Additionally, during stage 3, expression of certain markers, such as NGN3 expression, may be suppressed in properly specified endocrine cells. At stage 5, expression of NGN3 may be increased in properly specified endocrine cells. Properly specified endocrine cells may be monohormonal (e.g., INS only, G
Properly specified endocrine cells may be NKX6.1
Properly specified endocrine cells are monohormonal and may co-express other immature endocrine cell markers including NKX6.1 and PDX1. Properly specified endocrine cells are monohormonal and may co-express other immature endocrine cell markers including NKX6.1 and PDX1. Properly specified endocrine cells are monohormonal and may co-express other immature endocrine cell markers including NKX6.1 and PDX1.
In a preferred embodiment, the properly specified endocrine cells have at least 50% of single hormone expressing INS cells as a percentage of the total INS population. The properly specified endocrine cells may be CHGA+/INS+/NKX6.1+ (triple positive). In a preferred embodiment, more than about 25% of the cells in the cell population are CHGA+/TNS+/NKX6.1+ (triple positive). In a preferred embodiment, more than about 30% or more than 40% or more than 50% or more than 60% or more than 70% or more than 80% or more than 90% or more than 95% of the cells in the cell population are CHGA+/INS+/NKX6.1+ (triple positive).
Greater than or 100% are CHGA+/INS+/NKX6.1+ (triple positive).

The term "immature endocrine cells", in particular "immature beta cells", or equivalents thereof, includes at least INS, NKX6.1, PDX1, NEUROD, MNX1, NKX2.2,
It refers to cells derived from endocrine precursor/progenitor cells, pancreatic endoderm (PE) cells, pancreatic foregut cells, definitive endoderm cells, mesendoderm cells or any other endocrine cell precursors, including cells that express markers selected from the group consisting of MAFA, PAX4, SNAIL2, FOXA1 or FOXA2. The immature beta cells described herein preferably express INS, NKX6.1 and PDX1, and I
It is more preferable to co-express NS and NKX6.1.
The terms "immature pancreatic hormone-expressing cells" or "immature pancreatic islets" or equivalents thereof refer to, e.g., at least, unipotent immature beta cells, or pre-beta cells as described in FIG. 45, and do not include other immature cells, e.g., the term does not include immature alpha (glucagon) cells, or immature delta (somatostatin) cells, or immature epsilon (ghrelin) cells, or immature pancreatic polypeptide (PP) cells.

Many stem cell medium cultures or growth environments are contemplated in the embodiments described herein, including defined medium, conditioned medium, feeder-free medium, serum-free medium, and the like. As used herein, the term "growth environment" or "environment" or its equivalents is the environment in which undifferentiated or differentiated stem cells (e.g., primate embryonic stem cells) are grown in vitro. Characteristics of the environment include the medium in which the cells are cultured and, if present, a support structure (e.g., a substrate on a solid surface, etc.). Methods for culturing or maintaining pluripotent cells and/or for differentiating pluripotent cells are described in PCT/US2007/062755, entitled "Citation and Methods of Differentiating Pluripotent Cells."
OMPOSITIONS AND METHODS USEFUL FOR CULTURE
RING DIFFERENTIABLE CELLS, filed February 23, 2007; U.S. Patent Application Serial No. 11/993,399, entitled EMBRYONIC STEM CELL
CULTURE COMPOSITIONS AND METHODS OF USE
THEREOF, filed December 20, 2007; and U.S. patent application Ser. No. 11/875,0
No. 57, entitled "Methods and compositions for feed"
r-free pluripotent stem cell media conta
This is also described in the filing of US Pat. No. 6,313,323, filed Oct. 19, 2007.

The terms "essentially" and "substantially" mean the majority or de minimus of
By "cells" it is meant that the components or cells present in any cell population or cell culture, e.g., the immature beta cell cultures described herein, in reduced amounts, are "essentially or substantially cells" or are "immature beta cells that basally or substantially express INS, NKX6.1 and PDX1, and basally or substantially no NGN3 expression." Other examples include, but are not limited to, "essentially or substantially hES cells,""
"essentially or substantially definitive endoderm cells" or "essentially or substantially foregut endoderm cells"
"essentially or substantially PDX1-negative foregut endoderm cells,""essentially or substantially PDX1-positive pancreatic endoderm cells,""essentially or substantially pancreatic endocrine progenitor cells,""essentially or substantially pancreatic endocrine cells," and the like.

With respect to cells in a cell culture or cell population, the term "substantially free of" means
It means that a cell type identified as not being present in the cell culture or cell population is present in an amount of less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total number of cells present in the cell culture or cell population.

The terms "reduced serum" or "serum-free" or equivalents refer to a medium in which the cell culture grown contains reduced serum or is substantially free of serum or completely free of serum. Under certain culture conditions, the serum concentration is approximately 0% (v
For example, in some differentiation processes, the serum concentration in the medium ranges from less than about 0.05% (v/v), less than about 0.1% (v/v), less than about 0.2% (v/v),
(v/v), less than about 0.3% (v/v), less than about 0.4% (v/v), less than about 0.5% (v/v)
less than about 0.6% (v/v), less than about 0.7% (v/v), less than about 0.8% (v
less than about 0.9% (v/v), less than about 1% (v/v), less than about 2% (v/v)
, less than about 3% (v/v), less than about 4% (v/v), less than about 5% (v/v), less than about 6% (v/v)
The concentration of B27 supplement may range from about 0.1% (v/v) to about 20% (v/v). In some processes, the preprimitive streak cells are grown without serum or without serum replacement. In still other processes, the preprimitive streak cells are grown in the presence of B27. In such processes, the concentration of B27 supplement may range from about 0.1% (v/v) to about 20% (v/v).

In yet other processes, the cell culture is grown in the presence of B27. In such processes, the concentration of the B27 supplement may range from about 0.1% (v/v) to about 20% (v/v), or may be greater than about 20% (v/v). In certain processes, the concentration of B27 in the medium is about 0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v), about 0.6% (v/v), or greater than about 0.7% (v/v).
0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v), about 1% (
v/v), about 2% (v/v), about 3% (v/v), about 4% (v/v), about 5% (v/v)
, about 6% (v/v), about 7% (v/v), about 8% (v/v), about 9% (v/v), about 10
% (v/v), about 15% (v/v), or about 20% (v/v). Alternatively, the concentration of B27 supplement added can be measured as a multiple of the strength of a commercially available B27 stock solution. For example, B27 is available from Invitrogen (Carlsbad, Calif.) as a 50x stock solution. A sufficient amount of this stock solution is added to a sufficient volume of growth medium to produce a medium supplemented with the desired amount of B27. For example, 10 ml of 50x B27 stock solution is added to 90 ml of growth medium to produce 5x B27.
The resulting growth medium is supplemented with B27. The concentrations of B27 supplement in the medium are about 0.1x, about 0.2x, about 0.3x, about 0.4x, about 0.5x, about 0.6x, about 0.7x, about 0.9x, about 10x, about 11x, about 12x, about 13x, about 14x, about 15x, about 16x, about 17x, about 18x, about 19x, about 20x, about 21x, about 22x, about 23x, about 24x, about 25x, about 26x, about 27x, about 28x, about 29x, about 30x, about 31x, about 32x, about 33x, about 34x, about 35x, about 36x, about 37x, about 38x, about
7x, approximately 0.8x, approximately 0.9x, approximately 1x, approximately 1.1x, approximately 1.2x, approximately 1.3x, approximately 1.
4x, approximately 1.5x, approximately 1.6x, approximately 1.7x, approximately 1.8x, approximately 1.9x, approximately 2x, approximately 2.
5x, about 3x, about 3.5x, about 4x, about 4.5x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 11x, about 12x, about 13x, about 14x, about 15x, about 16x, about 1
It may be 7x, about 18x, about 19x, about 20x, and greater than about 20x.

In yet another embodiment, when the insulin level requirement for differentiation is low, B27 is not used since it contains high levels of insulin. For example, Applicants use 100x B27.
Insulin levels were determined in the 27 and ITS stocks, as well as in their respective 1× working stock solutions in RPMI.
Transsensitive insulin ELISA kits were used according to the manufacturer's instructions. Both kits were purchased from Life Technologies (Carlsbad, Calif.).
Unopened 100x and 50x B27 stocks and IT purchased from iFornia
The assay was performed on each of the S stocks. The results from the assay are shown in Table 3 below.
As shown in.

Table 3 shows approximately 3 μg/mL and 10 μg/mL in 1×B27 and 1×ITS, respectively.
It has been shown that there is 100/mL of insulin present. Furthermore, the insulin concentration in the 100x stock of ITS is consistent with the insulin concentration listed by the manufacturer.
Technologies does not provide insulin concentrations for 50xB27.
Therefore, there is no comparison with the insulin levels indicated by the manufacturer, but the above approximately 160
The insulin concentration in μg/mL was based on accurate measurements of 100× ITS stock performed simultaneously, and was determined based on the fact that both insulin and insulin-like growth factor (IGF) are PI3-
It has been shown to be a well-established agonist of kinase-dependent signaling,
It has been suggested that kinase inhibitors (e.g., LY294002) inhibit insulin/IGF effectors, thereby promoting definitive endoderm formation.
(2007) supra. See Figures 6A and 6B. In particular, SOX17 expression is reduced 4-fold at 1 μg/mL insulin. Insulin and/or IGF are not associated with KSR, FCS (fetal calf serum), FBS (fetal bovine serum), B2
7 and StemPro34, and may inhibit definitive endoderm differentiation under such culture conditions.
Ang et al. (2007) supra.

As used herein, the term "substance" refers to a substance, e.g., an agent, component, growth factor, differentiation factor, etc.
"Exogenous" or "exogenously added" compounds, with respect to culture or conditioned medium,
It refers to a compound that is added to a culture or medium to supplement any compound or growth factor that may or may not already be present in the culture or medium. For example, in some embodiments, the cell culture and/or cell population does not contain an exogenously added retinoid.

As used herein, "retinoid" refers to retinol, retinal or retinoic acid, as well as derivatives of any of these compounds. In a preferred embodiment, the retinoid is retinoic acid.

The term "FGF family growth factor,""fibroblast growth factor," or "member of the fibroblast growth factor family" refers to FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19 ...
F5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12
, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF
19. FGF selected from the group including FGF20, FGF21, FGF22 and FGF23
In some embodiments, "FGF family growth factor,""fibroblast growth factor" or "fibroblast growth factor family member" refers to any growth factor that has homology to and/or similar function to a known member of the fibroblast growth factor family.

The term "TGFβ superfamily growth factor" or "TGFβ superfamily ligand" or "TGFβ TGF-beta signaling pathway activator" or equivalents refer to TGF-beta 1, TGF-beta 2, TF-beta 3, GDF-15, G
DF-9, BMP-15, BMP-16, BMP-3, GDF-10, BMP-9, B
P-10, GDF-6, GDF-5, GDF-7, BMP-5, BMP-6, BMP-7
, BMP-8, BMP-2, BMP-4, GDF-3, GDF-1, GDF11, GDF
8, activin beta C, activin beta E, activin beta A and activin beta B, BMP-14, GDF-14, MIS, inhibin alpha, Lefty1, L
Refers to over 30 structurally related proteins, including the efty2, GDNF, neurturin, persephin and artemin subfamilies. Chang et al. (2002)
See Endocrine Rev. 23(6):787-823. These ligands are generally synthesized as prepropeptides of approximately 400-500 amino acids (aa), and the "TGFβ superfamily growth factors" or "TGFβ superfamily ligands" or "TGFβ TGF-beta signaling pathway activators" or equivalents may be full-length proteins or peptides derived from proteolysis thereof.

The term "ERBB ligand" refers to a ligand that binds to any one of the ErbB receptors, which in turn can form a dimer with another ErbB receptor or function as a monomer, thereby activating the tyrosine kinase activity of the ErbB partial monomer or dimer or heterodimeric receptor. Non-limiting examples of ErbB ligands include neuregulin-1; HRG-β, HRG-α, Neu differentiation factor (NRF), and the like.
DF), acetylcholine receptor induced activity (ARIA), glial growth factor 2 (GGF2
), and sensory and motor neuron derived factors (Sensory and Motor Neuron Derived Factors
splice variants and isoforms of Neuregulin-1, including but not limited to, Synaptic Neuron-Derived Factor (SMDF); Neuregulin-2; splice variants and isoforms of Neuregulin-2, including but not limited to, NRG2-β; Epiregulin; and Biregulin.
Examples include:

The term "expression" as used herein refers to the production of a material or substance as well as the level or amount of production of a material or substance. Thus, determining the expression of a particular marker refers to detecting the relative or absolute amount of the marker that is expressed or simply detecting whether the marker is present or absent.

The term "marker" as used herein refers to any molecule that can be observed or detected. For example, markers can include, but are not limited to, nucleic acids such as transcripts of a particular gene, polypeptide products of genes, non-gene product polypeptides, glycoproteins, carbohydrates, glycolipids, lipids, lipoproteins, or small molecules (e.g., molecules with a molecular weight of less than 10,000 amu).

For the majority of markers described herein, the official Human Genome Or
HUGO Gene No.
Such symbols, developed by the Menculation Committee, provide unique abbreviations for each named human gene and gene product, which those of skill in the art can readily recognize and associate with the corresponding unique human gene and/or protein sequences.

According to the HUGO nomenclature, the following gene symbols are defined as follows: GHRL-
Ghrelin; IAPP-islet amyloid polypeptide; INS-insulin; GCG-glucagon; ISL1-ISL1 transcription factor; PAX6-paired box gene 6; PAX4-
Paired box gene 4; NEUROG3-neurogenin 3 (NGN3); NKX2
-2-NKX2 transcription factor associated locus 2 (NKX2.2); NKX6-1-NKX6 transcription factor associated locus 1 (NKX6.1); IPF1-insulin promoter factor 1 (IPF1)
DX1);ONECUT1-Onecut domain, family member 1 (HNF6);
HLXB9 - homeobox B9 (HB9); TCF2 - transcription factor 2, liver (HNF1b
); FOXA1-Forkhead box A1; HGF-Hepatocyte growth factor; IGF1-Insulin-like growth factor 1; POU5F1-POU domain, class 5, transcription factor 1 (OCT
4); NANOG-Nanog homeobox; SOX2-SRY (sex determining region Y)-box 2; CDH1-cadherin 1, type 1, E-cadherin (ECAD); T-brac
hyury homolog (BRACH); FGF4-fibroblast growth factor 4; WNT3-wingless MMTV integration site family, member 3; SOX17-SRY (sex determining region Y)
-box 17; GSC-goosecoid; CER1-(cerberus 1, cysteine knot superfamily, homolog (CER); CXCR4-chemokine (C-X-C motif) receptor 4; FGF17-fibroblast growth factor 17; FOXA2-forkhead box A2; SOX7-SRY (sex determining region Y)-box 7; SOX1-SRY (sex determining region Y)-box 1; AFP-alpha-fetoprotein; SPARC-secreted protein, acidic, cysteine-rich (osteonectin); and THBD-thrombomodulin (TM), NCAM-neuronal cell adhesion molecule; SYP-synaptophysin; ZIC1-
Zic family member 1; NEF3-neurofilament 3 (NFM); SST-somatostatin; MAFA-v-maf musculoaponeurotic fibrosarcoma oncogene homolog A; MAFB-v-
maf-musculoaponeurotic fibrosarcoma oncogene homolog B; SYP-synaptophysin; CHGA-chromogranin A (parathyroid secretory protein 1); NGN3-neurogenin 3, NKX2
.2-NK2 homeobox 2; NKX6.1-NK6 homeobox 1; ID1-DN
A binding 1, GHRL - inhibitor of ghrelin/obestatin prepropeptide, GSK - glycogen synthase kinase, G6PC2 - glucose-6-phosphatase, UCN
3-Urocortin 3, PCSK1-Proprotein convertase subtilisin/kexin type 1, SLC30A8-Solute carrier family 30 (zinc transporter), member 8, and CA
DH1-E-cadherin. The terms fibroblast growth factor 7 (FGF7) and keratinocyte growth factor (KGF) are synonymous.

The progression from pluripotent cells to various multipotent and/or differentiated cells can be monitored by determining the relative expression of a gene, or gene marker, a characteristic of a particular cell, compared to the expression of a second or control gene, e.g., a housekeeping gene. In some processes, the expression of a particular marker is determined by detecting whether the marker is present or absent. Alternatively, the expression of a particular marker can be determined by measuring the level at which the marker is present in the cells of a cell culture or cell population. In such processes, the measurement of the expression of the marker can be qualitative or quantitative. One way to quantify the expression of a marker produced by a marker gene is by using quantitative PCR (Q-PCR). Methods for performing Q-PCR are well known in the art. Other methods known in the art can also be used to quantify marker gene expression. For example, the expression product of a marker gene can be detected by using an antibody specific for the marker gene product of interest.

Additional multiplexed assays and/or methodologies are available for analyzing large numbers of genes with high sensitivity and efficiency. For example, at least Nanostring (Se
Using the nCounter System provided by the National Institute of Advanced Industrial Science and Technology (Attleboro, WA, USA), users can simultaneously measure the expression levels of up to 800 genes using quantitative PCA.
The R system can be used to analyze the total RNA from any of the samples (e.g., stage 4 PEC and stage 7 endocrine precursor/precursor and endocrine cell cultures, respectively) using a 6100 Nucleic Acid Extractor (
The antibodies can be isolated (e.g., in triplicate) using a Nanostring nCounte™ PCR kit (Applied Biosystems; Foster City, Calif., USA) or a Nanostring nCounte™ PCR kit (Applied Biosystems; Foster City, Calif., USA).
Approximately 100 ng per reaction is required to quantify gene expression using the nCounter system. Also, instead of analog detection, the nCounter system uses digital detection, whereby each gene transcript is detected by a probe bound to a segment of DNA attached to a unique string of colored fluorophores (molecular barcode). Thus, the identity of the transcript depends only on the order of the fluorophores on the string, not on the intensity of the fluorophores. Second, the number of total transcripts in a sample is quantified by counting the total number of times a particular string of fluorophores (barcode) is detected. Furthermore, this method allows the detection of target mRNAs.
The system does not require amplification of A and therefore spans the biological range of expression, typically three orders of magnitude. Currently, with this system, as little as a 1.2-fold change in a single transcript at 20 copies (10 fM) per cell can be measured with statistical significance (p<0.05). For genes expressed at levels between 0.5 and 20 copies per cell, a 1.5-fold difference in expression level is detectable with the same level of confidence.

In some processes, certain populations of cells are indicated by high expression of the following genes compared to low expression of other genes, e.g., SOX17, SOX7, AFP, or TH
BD indicates extraembryonic endoderm, NODAL and/or FGF8 indicate the anterior primitive streak, brachyury, FGF4, SNAI1 and/or WNT3 indicate mesendoderm, CER, GSC, CXCR4, SOX17 and FOXA2 indicate definitive endoderm cells, SOX17, FOXA2, FOXA1, HNF1B and HNF4A indicate foregut endoderm (or PDX1-negative endoderm), PDX1, HNF6, SOX9
and PROX1, indicating PDX1-positive endoderm; PDX1, NKX6.1, PTF
A1, CPA and cMYC indicate pancreatic epithelium (PE or pancreatic precursor), and NGN
3. PAX4, ARX and NKX2.2 indicate endocrine precursor/progenitor cells, and INS
, GCG, GHRL, SST and PP indicate various endocrine cells, high MAFA gene expression relative to MAFB gene expression indicates insulin-secreting endocrine cells, and high expression of MAFB relative to MAFA gene expression indicates glucagon-secreting endocrine cells. Moreover, certain figures show only those "signature" or "important" markers typical of a particular cell type of a particular lineage. It will be well understood by those skilled in the art that other markers or genes are expressed but not shown or described, for example, genes that are constitutively expressed or expressed in all or most or the majority of cell types of a particular lineage or all lineages.

Methods for generating induced pluripotent stem (iPS) cells The embodiments described herein can be used to generate any one type of iPS cell or any one iPS cell.
There is also no limitation on the method of generating PS cells. Various methods exist, so the embodiments are not limiting or depend on the level of efficiency of generating iPS cells. The embodiments described herein apply to differentiation of iPS cells into endoderm lineage cells and uses thereof.

Research using certain nuclear reprogramming factors has made it possible to derive pluripotent stem cells or pluripotent-like stem cells from a patient's own somatic cells. These cells are also called induced pluripotent stem (iPS) cells.
Various iPS cell lines provided by the University of Toronto have been described. However, other iPS cell lines are contemplated by the present invention, such as those described in James Thomson et al., A1. US Patent Application Publication No. 20090047263
See, for example, International Publication No. WO 2005/80598, U.S. Patent Application Publication No. 20080233610, and International Publication No. WO 2008/11882. Thus, as used herein, "induced pluripotent stem (iPS) cells" refers to cells that have similar properties to other pluripotent stem cells, such as hES cells, hEG cells, pPS (primate pluripotent stem) cells, parthenogenetic cells, and the like.

Nuclear programming factors are described in U.S. Patent Application Publication No. 20090047263, International Publication No. WO 2009/023361, and the like.
US2005/80598, US20080233610 and International Publication WO2008/11882, and are used to induce reprogramming of differentiated cells without the use of eggs, embryos, or ES cells. The method for preparing iPS cells derived from somatic cells by using nuclear reprogramming factors similar to those used and described in the present invention is not particularly limited. In a preferred embodiment, the nuclear reprogramming factors are contacted with somatic cells in an environment in which somatic cells and induced pluripotent stem cells can proliferate. The advantage of certain embodiments described herein is that the reprogramming of iPS cells can be achieved without the use of eggs, embryos, or embryonic stem (ES) cells.
The advantage of this method is that induced pluripotent stem cells can be prepared by contacting nuclear reprogramming factors with somatic cells in the absence of cells. By using nuclear reprogramming factors, the nuclei of somatic cells can be reprogrammed to obtain iPS cells or "ES-like cells".

Pluripotent stem cells as described herein, whether hES cells or iPS cells, include, but are not limited to, alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60
;TRA-1-81;Tra-2-49/6E;ERas/ECAT5, E-cadherin;.beta.III-tubulin;.alpha.-smooth muscle actin (alpha.-SMA)
fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (N
at1); (ES cell-associated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2
;ECAT3;ECAT6;ECAT7;ECAT8;ECAT9;ECAT10;ECAT
AT15-1; ECAT15-2; Fthl17; Sall4; blastocyte transcription factor (
Utf1); Rex1; p53; G3PDH; telomerase including TERT; silent X genes; Dnmt3a; Dnmt3b; TRIM28; F-box-containing protein 15 (Fbx15); Nanog/ECAT4; Oct3/4; Sox2; Klf
4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; G
DF3; CYP25A1; developmental pluripotency-related 2
potency-associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1
), DPPA3/Stella; DPPA4, and the like. The present invention is not limited to the markers listed herein, and may include cell surface markers, antigens, and ESTs, RNA (microRNA and antisense RNA).
It is understood that the term encompasses markers such as markers of the present invention, including sequences thereof, DNA (including genes and cDNA) and other gene products, including portions thereof.

In one embodiment, the iPS cell lines used herein contain the following nuclear reprogramming factor genes: Oct family genes, Klf family genes, and Sox family genes.
Family genes. In one iPS cell line, the following three genes: Oct3/4
Three types of genes are provided: Oct family genes, Klf family genes, and Myc family genes, e.g., O
Other iPS cell line gene products of each of ct3/4, Klf4 and c-Myc were used, and it is therefore understood that nuclear reprogramming factors may or may not be associated with Myc family genes.

The nuclear reprogramming factors described herein and known in the art can be used to generate iPS cells from differentiated adult somatic cells and are not limited to the type of somatic cell that is reprogrammed, i.e., any kind of somatic cell can be reprogrammed or dedifferentiated. Since somatic cell reprogramming does not require an egg and/or embryo, the iPS cells can be mammalian cells, thus providing the opportunity to generate patient or disease specific pluripotent stem cells.

Adenovirus, plasmid, or Cre-loxP3 or piggy BAC
Induced pluripotent stem cells (iPSCs) using ablation of reprogramming factors using transposition
Viral, non-viral and non-integrating viral methods for generating the .
2, 945-949 (2008); Okita, K. et al., Science 322, 94
9-953 (2008); Kaji, K. et al., Nature 458, 771-775 (
2009); Soldner, F. et al., Cell 136, 964-977 (2009)
and Woltjen, K. et al., Nature 458, 766-770 (2009)
See also U.S. Patent Application Publication No. 2010000 to Mack, A. et al.
No. 3757 (published Jan. 7, 2010) and PCT/US2009/003666 to Shi et al.
See also 037429. However, the reprogramming efficiency of these methods is low (<
0.003%), despite excision, vector sequences may remain, thereby limiting their therapeutic application. For example, viral integration and overexpression of the above-mentioned transcription factors into the host genome has been linked to tumorigenesis, and residual transgene expression is a potential distinguishing feature between ES and iPS cells.
r, F. et al., Cell 136:964-977 (2009); Foster et al., Onc
Ogene 24:1491-1500 (2005); and Hochedlinger
See, K. et al., Cell 121:465-477 (2005).

In another embodiment of the present invention, the method for generating iPSCs includes an episomal vector derived from the Epstein-Barr virus. Yu, J. et al., Science 324
, 797-801 (2009) and U.S. Patent Application Publication No. 20 to Mack, A. et al.
See US Pat. No. 100003757, published Jan. 7, 2010. These methods require three separate plasmids carrying combinations of seven factors, including the oncogene SV40.

In another embodiment of the present invention, the method for generating iPSCs includes protein-based iPSCs from mouse cells and human fetal and neonatal cells.
et al., Cell Stem Cell 4, 381-384 (2009); and Kim,
See D. et al., Cell Stem Cell 4, 472-476 (2009). These methodologies are achieved using chemical treatments (e.g., valproic acid in the case of Zhou et al., 2009, supra) or multiple treatments (Kim et al., 2009, supra).

In another embodiment of the invention, minicircle vectors or plasmids, which are supercoiled DNA molecules lacking a bacterial origin of replication and an antibiotic resistance gene, can be used.
Chen, Z.-Y. et al., Mol. Ther. 8, 495-500 (2003);
Hen, Z.-Y. et al., Hum. Gene Ther. 16, 126-131 (20
05); and Jia, F. et al., Nature Methods Advances Pub.
See Litation Online 7 February 2010. These methodologies result in iPSCs with high transfection efficiency and longer ectopic expression due to the low activation of endogenous silencing mechanisms.

In yet another embodiment of the invention, iPS cells can be generated from human patients with various diseases, including diabetes, ALS, spinal muscular dystrophy, and Parkinson's patients. Maehr et al., PNAS USA 106(37):15768-73 (2009).
Dimos et al., Science 321:1218-21 (2008); Ebert et al., Nature 457:277-80 (2009); Park et al., Cell 134:
877-886 (2008); and Soldner et al., Cell 136:964-9
See, e.g., 77. At least one advantage of generating hIPS cells from patients with a particular disease is that the derived cells contain the genotype and cellular responses of the human disease. See also Table 4, which lists at least some of the existing human iPS cell lines. This information is provided in the literature and in, for example, the National Institutes of Health.
National Institutes of Health (NIH) Stem Cell Registry, the Human
Embryonic Stem Cell Registry and University
of Massachusetts Medical School, Worces
International St. in Boston, Massachusetts, USA
The data were derived from publicly available databases, including the<em>Cell Registry, which are regularly updated as cell lines become available and are registered.

In some embodiments of the compositions and methods described herein, other suitable cytochrome P450 antibodies may be used, including, but not limited to, CyT49, CyT212, CyT203, CyT25, (available at least at the time of the filing of this application from ViaCyte Inc., 3550 General Atomics Court, San Jose, CA).
(commercially available from San Diego, CA 92121), BGO1, BG02
and MEL1, and human pluripotent stem cells such as hESCs, including iPSC-482c.
7 and iPSC-603 (Cellular Dynamics International
iPSC-G4 (hereinafter "G4") and iPSC-B7 (hereinafter "B7") (Shional, Inc., Madison, Wisconsin)
Nya Yamanaka, Center for iPS Cell Research
induced pluripotent stem (iP h, commercially available from Kyoto University)
The use of a variety of differentiable primate pluripotent stem cells, including human iPS cells, is contemplated, and studies using these and other human iPS cells are described in U.S. patent application Ser. Nos. 12/765,714 and 13/761,078, both of which are entitled "CELL COMPOSITIONS, USE OF HUMAN IMPROVED PHARMACEUTICALLY ACTIVE STEROIDS, SUCCESSFULLY ACTIVELY ACTIVELY ACTIVELY ACTIVELY ACTIVELY ACTIVELY ACTIVELY ACTIVELY ACTIVELY ACTIVEly ...
FROM DEDIFFERENTIATED REPROGRAMMED CELL
S," filed April 22, 2010 and February 6, 2013, respectively. Certain of these human pluripotent stem cells are available from the National Institutes of Health, which is incorporated herein by reference in its entirety.
It is registered with international registration agencies such as the National Institutes of Health (NIH) and is listed in the NIH Human
They are listed in the Stem Cell Registry (e.g., CyT49 has the accession number 0041). Other available cell lines are listed at stemcells.nih.gov/.
They can also be found on the world wide web under research/registry.
Further cell lines, such as BG01 and BG01v, were obtained from Wisconsin
The cell lines described herein are commercially sold and distributed to third parties by WiCell® (catalog name, BG01) and ATCC (catalog number SCRC-2002), affiliates of the International Stem Cell Bank (WISC). Other cell lines described herein may not be registered or distributed by biological repositories such as WiCell® or ATCC, but such cell lines are publicly available, directly or indirectly, from principle researchers, laboratories and/or facilities. Requests for public disclosure of cell lines and reagents are routine for those of skill in the art, for example, in the life sciences. Typically, transfer of these cells or materials is via a standard material transfer agreement between the cell line or material owner and the recipient. These types of material transfers occur frequently, especially in the life science research environment. Indeed, Applicant has transferred CyT49 (2006), CyT203 (2005), Cyt212 (2006), Cyt312 (2007), Cyt49 (2008), Cyt512 (2009), Cyt612 (2010), Cyt712 (2011), Cyt812 (2012), Cyt912 (2013), Cyt101 (2014), Cyt111 (2015), Cyt121 (2016), Cyt1012 (2017), Cyt1013 (2018), Cyt1014 (2019), Cyt1015 (2010), Cyt1016 (2011), Cyt1017 (2012), Cyt1018 (2013), Cyt1019 (2014), Cyt1019 (2015), Cyt1019 (2016), Cyt1019 (2017), Cyt1019 (2018), Cyt1019 (2019), Cyt1019 (2013), Cyt1019 (201
09), CyT25 (2002), BG01 (2001), BG02 (2001), BG
Since the cells were derived and characterized, including BGOlv (2004), 03 (2001), and BGOlv (2004), cells have been routinely transferred through such agreements with commercial and non-commercial industrial partners and collaborators. The year in parentheses next to each cell line listed above indicates the year the cell line or material became publicly available and immortalized (e.g., a cell bank was created), and thus, no additional embryo destruction has been performed or required since these cell lines were established to make the compositions and practice the methods described herein.

Tables 4 and 5 are non-exhaustive lists of certain iPSCs and hESCs, respectively, that are available worldwide for research and/or commercial purposes and are suitable for use in the methods and compositions of the invention. The information in Tables 3 and 4 is taken from the literature and from, for example, Natl.
National Institutes of Health (NIH) Human Stem
Cell Registry, Human Embryonic Stem Cell
Registry and University of Massachusetts
Medical School, Worcester, Massachusetts, United States
The data were derived from publicly available databases, including the International Stem Cell Registry, located at:
It is updated regularly as cell lines become available and are registered.

Human iPSCs as described herein (at least iPSC-603 and iPSC-482)
-c7) is manufactured by Cellular Dynamics International, Inc.
(Madison, Wisconsin, USA).

Another advantage of using hIPS is that such hIPS cells are an immunologically compatible autologous cell population, allowing patient-specific cells to study the origin and progression of disease. Thus, the underlying causes of disease can be understood, which may provide insights that guide the development of preventative and therapeutic treatments against the disease.

Pluripotent human embryonic stem (hES) cells Some embodiments are directed to methods for deriving definitive endoderm cells and ultimately any endoderm lineage-derived cell type, including but not limited to foregut endoderm, pancreatic endoderm, endocrine precursor/precursor cells and/or pancreatic islet hormone-expressing cells, using human embryonic stem (hES) cells as starting material. These hES cells may originate from the morula, embryonic inner cell mass or from the gonadal ridges of an embryo. Human embryonic stem cells can be maintained in a pluripotent state without substantial differentiation in culture using methods known in the art. Such methods are described, for example, in U.S. Patent Nos. 5,453,357; 5,670,372; 5,690,926; 5,843,780; 6,200,806 and 6,251,671.

In some processes, pluripotent stem cells, such as hES cells, are maintained on a feeder layer. In such processes, any feeder layer that allows the pluripotent cells to be maintained in a pluripotent state can be used.
One commonly used feeder layer for pluripotent cell cultivation is a layer of mouse fibroblasts. More recently, human fibroblast feeder layers have been developed for use in the cultivation of pluripotent cells (U.S. Patent Application Publication No. 2002/0072117).
An alternative process allows for the maintenance of pluripotent cells without the use of a feeder layer.

The pluripotent cells described herein can be maintained in culture with or without serum, with or without extracellular matrix, with or without FGF. Some pluripotent cell maintenance procedures use serum replacements. These and other methods for culturing and differentiating pluripotent or multipotent cells are described in PCT/US200, respectively.
No. 7/062755, filed February 23, 2007, entitled COMPOSITIONS AND
METHODS FOR CULTURE DIFFERENTIAL CELLS
S and PCT/US2008/080516, filed October 20, 2008, entitled ME
THOD AND COMPOSITIONS FOR FEEDER-FREE P
LURIPOTENT STEM CELL MEDIA CONTAINING HU
It is described in MAN SERUM.

The invention described herein relates to all hES cell lines and at least some of the hES cell lines that are commercially available from the companies identified or available from WiCell on the world wide web at wicell.org.
/home/stem-cell-lines/order-stem-cell-lines
nes/obtain-stem-cell-lines.cmsx. This information is available from the literature and from, for example, the National Institutes of Health (NIH) Stem Cell Lines.
Cell Registry, Human Embryonic Stem Cell
Registry and University of Massachusetts
Medical School, Worcester, Massachusetts, United States
The data were derived from publicly available databases, including the International Stem Cell Registry, located at:
It is updated regularly as cell lines become available and are registered.
The International Stem Cell Registry contains at least 254 types of iPSCs.
Commercially available lines are listed, with 1211 hESC lines commercially available. Table 5 below is not an inclusive list of hESCs and iPSCs, but is an example of potentially available pluripotent stem cells.

Alternative Methods for Derivation of Pluripotent Stem Cells Methods exist for deriving pluripotent stem cells, such as mammalian ES cells, without destroying the embryo. Briefly, Advanced Cell Technology (Worces)
Three scientific papers were published by Johns Hopkins University Press, Massachusetts, USA, describing the derivation of mouse and human ES cells from single blastomeres while keeping the embryo intact and thus without causing its destruction. Later in 2005, Chung et al. first described a method for generating mouse ES cells from a single blastomere. Chung et al. (2006) Nature 439:216-219, 2005.
Published online on October 16, 2006. Chung et al. (2006)
described obtaining biopsies from embryos using micromanipulation techniques similar to those used for PGD (see page 217). At the time, Chung et al. (2006) co-cultured blastomere cell lines with other embryonic stem cells, although later developed methods made this unnecessary.
Ung et al. (2008), Human Embryonic Stem Cell Lin
es Generated without Embryo Destruction,
See Cell Stem Cell 2:113-117.

Preimplantation genetic diagnosis has been used to analyze genetic abnormalities in preimplantation embryos. The method is performed on early normally developing embryos (e.g., the 8-cell stage) at a time when the genetic input of the parents can be tested, by puncturing the zona pellucida and aspirating, extruding or dissecting one or two blastomeres through the opening. In PGD,
Genetic analysis is commonly used to analyze chromosomal abnormalities and single gene disorders using fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR), respectively. If no genetic abnormalities are found, the remaining intact embryo is then implanted into the female patient at a normal gestational age. Preimplantation genetic diagnosis techniques have already been developed in Sta.
Essen, C. et al. (2004), Comparison of blastocysts
transfer with or without preimplantation
Genetic diagnosis for aneuploy screen
ing in couples with advanced material agency
e: a prospective randomized controlled tr
ial, Hum. Reprod. 19:2849-2858 and Monni et al.
004) Preimplantation genetic diagnosis for
r beta-thalassemia: the Sardinian experience
Prenat Diagn 24:949-954. Thus, Chung et al. (2006) simply described methods available for extracting blastomeres from early embryos without destroying the embryo.

Furthermore, in August 2006, Klimanskaya et al., second authors of the Chung et al. (2006) publication, also from Advanced Cell Technology, reported that a similar PGD technique, although less efficient (hES cell lines were generated from only 2% of isolated blastomeres), allowed human ES cell lines to be derived from single blastomeres.
(2006) described a procedure that was demonstrated to be not significantly different from that originally described above.
Nick stem cell lines derived from single
blastomeres, Nature 444, 481-485.
Imanskaya et al. co-cultured the newly derived hES cell line with other ES cells,
This is something Chung et al. (2006) said could be important. They wrote, "it is unclear whether the success
ss of the ES co-culture system in [his]
study is attributeable to substances secret
ed by the ES cells or if cell-cell con
tact is required." Chung et al. (2006), supra, at 21
See page 8, right column. However, later in 2008, Chung et al., also cited above, demonstrated that culturing isolated blastomeres in medium with laminin enhanced their ability to give rise to hESCs, so that hES cell lines did not require any co-culture with ES cells. Chung et al. (2008), Human Embryonic Stem Cells, vol.
em Cell Lines Generated without Embryo D
, Cell Stem Cell (2): 113-117, 116
See, p. 1, published online Jan. 10, 2008. Moreover, the hES cells thus obtained have the same properties as other human pluripotent stem cells, including hES cells, including the ability to be maintained in an undifferentiated state for over six months, and have the same characteristics as Oct-4, SSEA-3, S
The mice showed normal karyotype and expression of markers of pluripotency, including SEA-4, TRA-1-60, TRA-1-81, Nanog and alkaline phosphatase, and showed in vitro
They can differentiate and form derivatives of all three embryonic germ layers both in vitro and in vivo in the form of teratomas.

Thus, if it is not critical to co-cultivate a single blastomere with other ES cells, as originally postulated by Chung et al. (2006), simply culturing the single blastomere with laminin, which is a common component of many extracellular matrices (commercially available and/or made, for example, by lysing feeder cells), is commonly used, and was known to grow mammalian cells, and the like, was known and available at the time Chung's 2006 publication first demonstrated the derivation of mouse ES cell lines from single blastomeres. Thus, it is entirely possible that one skilled in the art could take the methodology of Chung et al. (2006) supra and use the available knowledge previously described by Straessen et al. (2004) supra to derive hES cell lines from single blastomeres while preserving or preventing the destruction of the embryo.

Aggregate Suspensions of Pluripotent Stem Cells and Cells Derived from Pluripotent Stem Cells In contrast to previously known tissue engineering methods based on seeding individual cells onto polymer scaffolds, matrices and/or gels, the embodiments described herein may use cell aggregate suspensions formed from pluripotent stem cells, single cell suspensions or differentiated single cell suspensions derived therefrom. Processing and/or Processing of Stem Cell Aggregate Suspensions
The method of production and differentiation of the cells are described in PCT/US2007/062755, 20
Filed on February 23, 2007, entitled COMPOSITIONS AND METHODS FOR
R CULTURING DIFFERENTIAL CELLS, now U.S. Pat.
, 211,699 and 8,415,158; and PCT/US2008/0
No. 80516, filed October 20, 2008, entitled METHODS AND COMPOS
ITIONS FOR FEEDER-FREE PLURIPOTENT STEM
CELL MEDIA CONTAINING HUMAN SERUM, now U.S. Patent No. 8,334,138, and Schulz T. et al. (2012), supra.

Embodiments described herein relate to a method for generating pluripotent cell aggregates in suspension from a pluripotent adherent culture by culturing pluripotent cells in adherent growth culture conditions that allow for expansion in an undifferentiated state, dissociating the adherent pluripotent cell culture into a single cell suspension culture, and contacting the single cell suspension culture with first differentiation culture conditions that allow for the formation of hES-derived cell aggregates in suspension by agitating the single cell suspension culture for a period of time during which the single cell suspension culture forms pluripotent-derived cell aggregates in suspension, thereby generating pluripotent-derived cell aggregates in suspension. In a preferred embodiment, agitation of the single cell suspension culture is performed by rotating at about 80 rpm to 160 rpm. Certain other embodiments described herein use a rho-kinase inhibitor to promote aggregation, growth, proliferation, expansion and/or cell mass of pluripotent stem cells.

The phrases "substantially uniform" or "substantially uniform in size and shape" or equivalents refer to the spread of uniformity of the aggregates being about 20% or less. In another embodiment, the spread of uniformity of the aggregates is about 15% or less, about 10% or less, or about 5% or less.

In yet another embodiment, the hES cell aggregate suspension is cultured in a medium that is substantially free of serum.
Additionally, they were cultured in the absence of exogenously added fibroblast growth factor (FGF), which means that they were cultured in medium without serum but containing exogenously added growth factors, including FGF.
U.S. Patent No. 7,005 to Thomson, J., which requires culturing ES cells;
In some embodiments, the iPS cell aggregate suspension is cultured in a medium that is substantially free of serum and/or further contains exogenously added fibroblast growth factors (
The cells are cultured in the absence of FGF.

While the exact number of cells per aggregate is not critical, one of skill in the art will appreciate that the size of each aggregate (and therefore the number of cells per aggregate) is limited by the maximum diffusion of oxygen and nutrients to the central cell, and that this number may also vary depending on the cell type and the nutrient requirements of that cell type. Cell aggregates may contain a minimum number of cells per aggregate (e.g., two or three cells) or may contain hundreds or thousands of cells per aggregate. Typically, cell aggregates contain hundreds to thousands of cells per aggregate. For purposes of the present invention, cell aggregates are generally from about 50 microns to about 600 microns in size, although depending on the cell type, the size may be below or above this range. In one embodiment, the cell aggregates are from about 50 microns to about 250 microns in size, or from about 75-200 microns in size, with about 100-150 microns being preferred.

Yet another method describes the generation of embryoid bodies (EBs). As used herein, the terms "embryoid bodies", "aggregate bodies" or equivalents refer to aggregates of differentiated and undifferentiated cells that arise when ES cells are overgrown in monolayer cultures or maintained in suspension cultures in undefined media or differentiated into multiple germ layer tissues by non-directed protocols. That is, EBs are not formed from single cell suspensions of pluripotent stem cells as described herein, nor are EBs formed from adherent cultures of pluripotent-derived multipotent cells. These features alone clearly distinguish the present invention from embryoid bodies.

In contrast to embryoid bodies, which are mixtures of differentiated and undifferentiated cells, generally consisting of cells derived from several germ layers and which undergo random differentiation, the cell aggregates described herein are essentially or substantially homogeneous cells and exist as aggregates of pluripotent, multipotent, bipotent, or unipotent types of cells, e.g., embryonic cells, definitive endoderm, foregut endoderm, PDX1-positive pancreatic endoderm, pancreatic endocrine cells, etc.

The methods described herein, for example, to Bodnar et al.
There is no need to first coat the culture vessel with an extracellular matrix as described in U.S. Patent No. 6,800,480 assigned to the U.S. Patent No. 6,800,480 of the same assignee as the present invention. In some embodiments described herein, iPS cells can be cultured in the same manner as other pluripotent stem cells, such as hES cells and iPS cells, are cultured using soluble human serum, substantially as described in Applicant's U.S. Patent No. 8,334, 138; and Schulz T. et al. (2012) supra.

The methods described herein are directed to Thomson, J., and Wiscon
No exogenously added fibroblast growth factor (FGF) is required, provided from a source other than simply a fibroblast feeder layer, as described in US Pat. No. 7,005,252, assigned to the sin Alumni Research Foundation (WARF).

Pluripotent and Differentiated Cell Compositions The cell compositions produced by the methods described herein include pluripotent stem cells, preprimitive streak, mesendoderm, definitive endoderm, foregut endoderm, PDX1 positive foregut endoderm, PDX1 positive pancreatic endoderm or PDX1/NKX6.1 co-positive pancreatic endoderm, endocrine precursors/precursors or NGN3/N
KX2.2 co-positive endocrine precursors/precursors and hormone-secreting endocrine cells or INS
, GCG, GHRL, SST, PP single positive endocrine cells, wherein at least about 5-90% of the cells in the culture are generated anterior primitive streak, mesendoderm, definitive endoderm, foregut endoderm, PDX1 positive foregut endoderm, PDX1 positive pancreatic endoderm or PDX1/NKX6.
1 co-positive pancreatic endoderm, endocrine precursors/precursors or NGN3/NKX2.2 co-positive endocrine precursors/precursors, and hormone-secreting endocrine cells or INS, GCG, GHRL,
SST and PP are single positive endocrine cells.

Some embodiments described herein provide for the production of both pluripotent cells, such as stem cells and iPS cells, and multipotent cells, such as preprimitive streak, mesendoderm, or definitive endoderm, as well as PDX1-positive foregut endoderm, PDX1-positive pancreatic endoderm, or PDX1/NKX6.1 co-positive pancreatic endoderm,
The present invention relates to compositions such as cell populations and cell cultures that contain endocrine precursors/precursors or NGN3/NKX2.2 co-positive endocrine precursors/precursors, and more differentiated but potentially multipotent cells such as hormone-secreting endocrine cells or INS, GCG, GHRL, SST, PP single positive endocrine cells. For example, the methods described herein can be used to generate compositions that contain a mixture of pluripotent stem cells and other multipotent or differentiated cells. In some embodiments, compositions are generated that contain at least about 5 multipotent or differentiated cells for every about 95 pluripotent cells. In other embodiments, compositions are generated that contain at least about 95 multipotent or differentiated cells for every about 5 pluripotent cells.
Additionally, compositions comprising other ratios of multipotent or differentiated cells to pluripotent cells are contemplated, for example, compositions comprising at least about 1 multipotent or differentiated cell for about every 1,000,000 pluripotent cells, compositions comprising at least about 1 multipotent or differentiated cell for about every 100,000 pluripotent cells, compositions comprising at least about 1 multipotent or differentiated cell for about every 10,000 pluripotent cells, compositions comprising at least about 1 multipotent or differentiated cell for about every 1000 pluripotent cells, compositions comprising at least about 1 multipotent or differentiated cell for about every 500 pluripotent cells, compositions comprising at least about 1 multipotent or differentiated cell for about every 10
A composition comprising at least about 1 multipotent or differentiated cell for every 0 pluripotent cells;
Compositions comprising at least about 1 multipotent or differentiated cell for about every 10 pluripotent cells, compositions comprising at least about 1 multipotent or differentiated cell for about every 5 pluripotent cells, and up to about 1 pluripotent cell, and compositions comprising at least about 1 multipotent or differentiated cell for about every 1 pluripotent cell.
Compositions comprising millions of multipotent or differentiated cells are contemplated.

Some embodiments described herein relate to cell cultures or cell populations comprising at least about 5% multipotent or differentiated cells to at least about 99% multipotent or differentiated cells. In some embodiments, the cell cultures or cell populations comprise mammalian cells. In preferred embodiments, the cell cultures or cell populations comprise human cells. For example,
Certain embodiments provide a cell culture comprising human cells, comprising at least about 5% of the human cells.
The present invention relates to cell cultures in which from about 99% to at least about 99% are multipotent or differentiated cells.
Other embodiments relate to cell cultures comprising human cells, wherein at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more than 99% of the human cells are multipotent or differentiated cells. In embodiments where the cell culture or cell population comprises human feeder cells, the above percentages are calculated without consideration of the human feeder cells in the cell culture.

The compositions and methods described herein have several useful features. For example, cell cultures and cell populations including pluripotent cells, such as preprimitive streak cells and/or mesendoderm cells, and methods for generating such cell cultures and cell populations are useful for modeling early human development. In addition, the compositions and methods described herein may also serve as therapeutic interventions for pathologies such as diabetes mellitus. For example, preprimitive streak cells and/or mesendoderm cells serve as a source for only a limited number of tissues, and therefore can be used in the generation of pure tissue or cell types. In some processes for generating preprimitive streak cells, the pluripotent cells used as starting material are pluripotent stem cells, such as hESCs, which are pluripotent stem cells.
ES cells, hEG cells or iPS cells.

Trophectoderm Cells The methods described herein can be used to generate compositions comprising trophectoderm cells, substantially free of other cell types. In some embodiments described herein, the trophectoderm cell populations or cell cultures generated by the methods described herein contain HAND1, E
omes, MASH2, ESXL1, HCG, KRT18, PSG3, SFXN5, DL
X3, PSX1, ETS2, and ERBB genes in a non-trophectoderm cell or cell population.
ESXL1, HCG, KRT18, PSG3, SFXN5, DLX3, PSX1, ETS
2, and is substantially higher compared to the expression levels of ERBB.

Preprimitive streak cells The methods described herein can be used to generate compositions comprising preprimitive streak cells, substantially free of other cell types. In some embodiments described herein, the preprimitive streak cell populations or cell cultures generated by the methods described herein express the FGF8 marker gene and/or the NODAL marker gene as follows: BRACHURY low, FGF4 low,
Preprimitive streak cells and methods of generating preprimitive streak cells are described in Applicant's U.S. Pat. No. 7,958,585, PREPRIMITIVE STREAK CELLS AND METHODS FOR PRODUCING THE SAME.
This is described in detail in TREAK AND MESENDODERM CELLS, published on July 26, 2011.

The methods described herein can be used to generate compositions comprising extraembryonic cells, substantially free of other cell types. Primitive endoderm cells, proximal endoderm cells and distal endoderm cells are extraembryonic cells. Primitive endoderm cells give rise to proximal endoderm cells and distal endoderm cells.
Proximal endoderm cells are endoderm cells that form part of the yolk sac. Proximal endoderm cells function in both nutrient uptake and transport. Distal endoderm cells are in close proximity to an extraembryonic tissue known as Reichert's membrane. One of the roles of distal endoderm cells is the production of components of the basement membrane. Taken together, proximal and distal endoderm cells form what is often referred to as extraembryonic endoderm. As the name suggests, extraembryonic endoderm cells do not give rise to embryonic structures that form during development. In contrast, the definitive endoderm cells and other endoderm lineage or pancreatic lineage cells described herein are embryonic or derived from embryonic cells, and give rise to tissues derived from the gut tube that form during embryonic development. In some embodiments described herein, the extraembryonic cell populations or cell cultures generated by the methods described herein have substantially higher expression of a marker selected from the group including SOX7 gene, SOX17 gene, THBD gene, SPARC gene, DAB1 gene, FTNF4alpha gene or AFP gene, at least compared to the expression levels of SOX7, SOX17, THBD, SPARC, DAB1, or AFP that are not expressed in other cell types or cell populations, e.g., definitive endoderm.

Mesendoderm Cells The methods described herein can be used to generate compositions comprising mesendoderm cells, substantially free of other cell types. In some embodiments described herein, the mesendoderm cell populations or cell cultures generated by the methods described herein contain FGF4, SNAI1
, MIXL1 and/or WNT3 marker genes, SOX17 low, CXCR4 low, FOXA2 low, SOX7 low,
Mesendoderm cells and methods for generating mesendoderm cells are described in Applicant's U.S. Pat. No. 7,958,585, PREPRIMITIV
E STREAK AND MESENDODERM CELLS, July 26, 2011
It's explained in detail in the daily issue.

Screening Method In some embodiments, the screening method is used to obtain a certain cell population including pluripotent cells, multipotent cells and/or differentiated cells, such as human pluripotent stem cells, induced pluripotent stem cells, preprimitive streak cells, mesendoderm cells, definitive endoderm cells, foregut endoderm or PDX1-negative foregut endoderm cells, PDX1-positive foregut endoderm or PDX1-positive pancreatic endoderm cells or pancreatic progenitor cells, endocrine precursor/precursor cells, and/or endocrine cells. A candidate differentiation factor is then provided to the cell population. At a first time point, before or at about the same time as providing the candidate differentiation factor, the expression of the marker is determined. Alternatively, the expression of the marker can be determined after providing the candidate differentiation factor. At a second time point, after the first time point and after the step of providing the candidate differentiation factor to the cell population, the expression of the same marker is determined again. Whether the candidate differentiation factor can promote the differentiation of pancreatic progenitor cells is determined by comparing the expression of the marker at the first time point with the expression of the marker at the second time point. If expression of the marker at the second time point is increased or decreased relative to expression of the marker at the first time point, then the candidate differentiation factor is capable of promoting differentiation of pancreatic progenitor cells.

In some embodiments of the screening methods described herein, human definitive endoderm, PDX
The present invention utilizes a cell population or cell culture comprising PDX-1-negative foregut endoderm, PDX-1-positive foregut endoderm, PDX-1-positive pancreatic endoderm, or pancreatic precursor or endocrine precursor/progenitor cells. For example, the cell population can be a substantially purified population of PDX-1-positive pancreatic endoderm or pancreatic progenitor cells. For example, the cell population can be an enriched population of human pancreatic progenitor cells,
At least about 50%-97% of the human cells in the cell population are human pancreatic progenitor cells, with the remainder consisting of endocrine precursors/precursors or endocrine cells and other cell types. Enrichment of the pancreatic progenitor population is described in applicant's U.S. patent application Ser. No. 12/107,020, entitled METHODS FOR USE IN HIGH-TEMPERATURE HUMAN CARDIAC SYSTEMS.
R PURIFYING ENDODERM AND PANCREATIC ENDO
DERM CELLS DERIVED FROM HUMAN EMBRYONICS
STEM CELLS, filed April 21, 2008, now U.S. Pat. No. 8,338,177.
0 and the corresponding publication Kelly et al. (2011), supra.

In some embodiments of the screening methods described herein, the cell population is a candidate (test)
The cells are contacted with or otherwise provided with a candidate (test) differentiation factor. Candidate differentiation factors may include any molecule that may have the potential to promote differentiation of any of the above cells, e.g., human pancreatic progenitor cells. In some embodiments described herein, the candidate differentiation factor is
The candidate differentiation factors include molecules known to be differentiation factors for one or more types of cells. In alternative embodiments, the candidate differentiation factors include molecules not known to promote cell differentiation. In preferred embodiments, the candidate differentiation factors include molecules not known to promote differentiation of human pancreatic progenitor cells. Screening for factors that differentiate definitive endoderm can be performed, for example, as described in applicant's U.S. patent application Ser. No. 12/093,590, entitled MARKERS OF DISEASE.
This is described in detail in EFINITIVE ENDODERM, filed July 21, 2008.

In addition to determining the expression of at least one marker at a first time point, some embodiments of the screening methods described herein contemplate determining the expression of at least one marker at a second time point that is subsequent to the first time point and subsequent to providing the cell population with the candidate differentiation factor. In such embodiments, the expression of the same marker is determined at both the first and second time points. In some embodiments, the expression of multiple markers is determined at both the first and second time points. In such embodiments, the expression of the same multiple markers is determined at both the first and second time points. In some embodiments, the expression of the marker is determined at multiple time points, each subsequent to the first time point and each subsequent to providing the cell population with the candidate differentiation factor. In certain embodiments, the expression of the marker is determined by Q-PCR. In other embodiments, the expression of the marker is determined by immunocytochemistry.

In certain embodiments of the screening methods described herein, the markers whose expression is determined at the first and second time points are markers associated with the differentiation of pancreatic progenitor cells into cells that are precursors of the terminally differentiated cells that make up pancreatic islet tissue, including immature pancreatic islet hormone-expressing cells.

In some embodiments of the screening methods described herein, a sufficient time is allowed to elapse between providing the cell population with the candidate differentiation factor and determining expression of the marker at a second time point. The sufficient time between providing the cell population with the candidate differentiation factor and determining expression of the marker at a second time point may be as little as about 1 hour to as long as about 10 days. In some embodiments, the expression of at least one marker is determined multiple times after providing the cell population with the candidate differentiation factor. In some embodiments, a sufficient time is at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 16 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 1 week,
At least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks,
At least about 6 weeks, at least about 7 weeks, or at least about 8 weeks.

In some embodiments of the methods described herein, the method further determines whether the expression of the marker at the second time point is increased or decreased compared to the expression of the marker at the first time point. An increase or decrease in the expression of at least one marker indicates that the candidate differentiation factor can promote the differentiation of endocrine precursor/precursor cells. Similarly, when the expression of multiple markers is determined, the method further determines whether the expression of the multiple markers at the second time point is increased or decreased compared to the expression of the multiple markers at the first time point. The increase or decrease in the expression of the marker can be determined by measuring or otherwise evaluating the amount, level or activity of the marker in the cell population at the first and second time points. Such determination may be relative to other markers, e.g., housekeeping gene expression, or may be absolute. In certain embodiments in which the expression of the marker at the second time point is increased compared to the first time point,
The amount of increase is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or at least more than 100-fold. In some embodiments, the amount of increase is less than 2-fold. In embodiments in which expression of the marker at the second time point is decreased compared to the first time point, the amount of decrease is at least about 2-fold, at least about 5-fold, at least about 10 ...
at least about 1/20, at least about 1/30, at least about 1/40,
At least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or at least less than about 100-fold. In some embodiments, the amount of reduction is no more than a factor of 2.

Monitoring the generation of multipotent or differentiated cells The progression of pluripotent cells to multipotent cells, and from multipotent cells to more multipotent or differentiated cells, such as pancreatic precursors or hormone endocrine cells, can be monitored by determining the expression of markers characteristic of a particular cell, including genetic and phenotypic markers, such as expression of islet hormones and the processing of proinsulin to insulin and C-peptide in endocrine cells. In some processes, the expression of a particular marker is determined by detecting the presence or absence of the marker.
Alternatively, expression of a particular marker can be determined by measuring the level at which the marker is present in the cells of the cell culture or cell population. For example, in certain processes, expression of markers characteristic of immature pancreatic islet hormone-expressing cells, as well as pluripotent cells,
The lack of significant expression of markers characteristic of definitive endoderm, foregut endoderm, PDX1-positive foregut endoderm, endocrine precursors/precursors, extraembryonic endoderm, mesoderm, ectoderm, mature pancreatic islet hormone-expressing cells and/or other cell types is determined.

Marker expression can be measured using qualitative or semi-quantitative techniques such as blot transfer and immunocytochemistry, as described in connection with monitoring the generation of other less differentiated cell types of the definitive endoderm lineage. Alternatively, marker expression can be accurately quantified by using techniques such as Q-PCR or Nanostring's nCounter or related high throughput multiplex technologies that can analyze and assay hundreds or more markers simultaneously. Additionally,
At the polypeptide level, it will be appreciated that many of the markers for pancreatic islet hormone-expressing cells are secreted proteins. Thus, techniques for measuring extracellular marker content, such as ELISA, are available.

In other embodiments, immunohistochemistry is used to detect proteins expressed by the above genes. In yet other embodiments, Q-PCR can be used in conjunction with immunohistochemistry or flow cytometry techniques to effectively and accurately characterize and identify cell types and determine both the amount and relative proportion of such markers in the cell type of interest. In one embodiment, Q-PCR can quantify the level of RNA expression in cell cultures containing a mixed population of cells. However, Q-PCR cannot provide or qualify whether markers or proteins of interest are co-expressed in the same cell. In another embodiment, Q-PCR is used in conjunction with flow cytometry methods to characterize and identify cell types. Thus, by using the methods described herein in combination with, for example, those described above, complete characterization and identification of various cell types, including endoderm lineage cells, can be achieved and demonstrated.

For example, in a preferred embodiment, the pancreatic progenitor or pancreatic endoderm or PDX-1 positive pancreatic endoderm is at least PDZ-1 positive as demonstrated by Q-PCR and/or ICC.
PDX1 positive expressing cells express PDX1, Nkx6.1, PTF1A, CPA and/or cMYC, but such cells must co-express at least PDX1 and Nkx6.1 as demonstrated by ICC and not express other markers, including SOX17, CXCR4, or CER, to be identified as PDX1 positive expressing cells.
To adequately identify mature hormone-secreting pancreatic cells in vivo, for example, ICC demonstrates that in insulin-secreting cells, C-peptide (the product of proper processing of proinsulin in mature and functional β-cells) and insulin are co-expressed.

In addition, other methods known in the art can be used to quantify marker gene expression. For example, expression of marker gene products can be detected by using antibodies specific to the marker gene products of interest (e.g., Western blot, flow cytometry analysis, etc.). In certain processes, expression of marker genes specific to hES-derived cells as well as the lack of significant expression of marker genes specific to hES-derived cells. Further methods for characterizing and identifying hES-derived cell types are described in the related applications listed above.

Summary of In Vivo Generation of PDX1-Positive Pancreatic Endoderm (Stages 1-4) and Insulin Production Methods for generating certain endoderm-lineage cells and pancreatic endoderm-lineage cells are provided herein and are described in U.S. Pat. Nos. 7,534,608; 7,695,965; and 7,993,920, entitled METHODS OF PRODUCING PANCREATIC ENDODERM.
CREATIC HORMONES; and U.S. Patent No. 8,129,182, entitled
DOCRINE PROGENITOR/PRECURSOR CELLS, PANCR
EATIC HORMONE-EXPRESSING CELLS AND METHO
These are discussed elsewhere in related applications, such as DS OF PRODUCTION.
Also see Figures 42, 43 and 44.

Briefly, the directed differentiation method of the present invention for pluripotent stem cells, e.g., hES cells and iPS cells, can be described in at least four or five or six or seven stages depending on the desired end stage cell culture (PEC cells or endocrine cells). Stage 1 is the generation of definitive endoderm from pluripotent stem cells, which takes about 2-5 days, preferably 2 or 3 days. The pluripotent stem cells are cultured in RPMI, TGFβ superfamily member growth factors, such as activin A, activin B, GDF-8 or GDF-11, etc.
100 ng/mL), a Wnt family member or a Wnt pathway activator, e.g., W
The cells are suspended in a medium containing a rho kinase or ROCK inhibitor, such as Y-27632 (10 μM) to enhance growth and/or survival and/or proliferation and/or cell-cell adhesion, such as nt3a (25 ng/mL). After about 24 hours, the medium is changed to RPMI containing serum, such as 0.2% FBS, and a TGFβ superfamily member growth factor, such as activin A, activin B, GDF-8 or GDF-1.
The medium is then replaced with medium containing activin/Wnt3a (100 ng/mL) or a rho-kinase or ROCK inhibitor for an additional 24 hours (day 1) to 48 hours (day 2). Alternatively, after approximately 24 hours in medium containing activin/Wnt3a, the cells are cultured for the next 24 hours in medium containing activin alone (i.e., the medium does not contain Wnt3a). Importantly, the generation of definitive endoderm requires cell culture conditions with low serum content, and therefore low insulin or insulin-like growth factor content. McLean et al. (2007) S
See TEM Cells 25:29-38. McLean et al. have also shown that contacting hES cells at stage 1 with insulin at a concentration of as little as 0.2 μg/mL can be detrimental to the generation of definitive endoderm.
Stage 1 differentiation of pluripotent cells to definitive endoderm is substantially as described herein and in D'Amour's
(2005). For example, at least one of the methods described in Agarwal et al., Ef
Differentiation of Functional He
Patocytes from human embryonic stem cells
s, Stem Cells (2008) 26:1117-1127; Borowiak et al., Small Molecules Efficiently Direct Endo
Dermal Differentiation of Mouse and Human
Embryonic Stem Cells, (2009) Cell Stem Cell
4:348-358; and Brunner et al., Distinct DNA met.
Hydration patterns characterize different
induced human embryonic stem cells and development
eloping human fetal liver, (2009) Genome R
es. 19:1044-1056. Proper differentiation, specification, characterization and identification of definitive endoderm is necessary to derive other endoderm lineage cells. Definitive endoderm cells at this stage co-express SOX17 and HNF3β (FOXA2) and at least
F4 alpha, HNF6, PDX1, SOX6, PROX1, PTF1A, CPA, cM
YC, NKX6.1, NGN3, PAX3, ARX, NKX2.2, INS, GSC, G
There is no appreciable expression of HRL, SST, or PP.
The absence of NF4alpha expression is consistent with at least the findings of Duncan et al. (1994), "E
Expression of transcription factor FTNF-4
In the extraembryonic endoderm, gut, and
d nephrogenic tissue of the developing m
Ouse Embryo: FTNF-4 is a marker for priming
ary endoderm in the implanting blastocysts
t", Proc. Natl. Acad. Sci., 91:7598-7602 and Si-
Tayeb et al. (2010), "Highly Efficient Generation
of human hepatocyte-like cells from In
"Induced Pluripotent Stem Cells," Hepatology
51:297-305.

In stage 2, definitive endoderm cell cultures are obtained from stage 1 and the suspension cultures are cultured in an IT
Foregut endoderm or PDX1-negative foregut endoderm is generated by incubation for 24 hours (days 2-3) with RPMI with low serum levels such as 0.2% FBS, 25 ng KGF (or FGF7), or ROCK inhibitor in a 1:1000 dilution of S. After 24 hours (days 3-4), the medium is replaced with the same medium without TGFβ inhibitor but instead still containing ROCK inhibitor to enhance cell growth, survival and proliferation for another 24 hours (days 4-5) to 48 hours (day 6). A critical step to properly specify foregut endoderm is the removal of TGFβ family growth factors. Therefore, a TGFβ inhibitor such as 2.5 μM TGFβ inhibitor number 4 or 5 μM SB431542, a specific inhibitor of the TGFβ type I receptor, activin receptor-like kinase (ALK), can be added to the stage 2 cell culture. Foregut endoderm or PDX1-negative foregut endoderm cells generated at stage 2 were identified as SOX17, HNF1β, and HN
At least SOX17 and HNF3β (FOXA2), which co-express F4alpha and are hallmarks of definitive endoderm, PDX1-positive pancreatic endoderm or pancreatic progenitor cells or endocrine precursors/precursors, and generally, multihormonal cells, are also expressed by HNF6, PDX1, SOX6, P
ROX1, PTF1A, CPA, cMYC, NKX6.1, NGN3, PAX3, ARX
, NKX2.2, INS, GSC, GHRL, SST, or PP were not appreciably co-expressed.

For stage 3 of PEC generation (days 5-8), foregut endoderm cell cultures were obtained from stage 2 and incubated with 1% B27, 0.25 μM KAAD cyclopamine, 0.2 μM retinoid such as retinoic acid (RA) or a retinoic acid analog such as 3 nM TTNPB (or CTT3, which is a combination of KAAD cyclopamine and TTNPB), and 50 ng/mL
in Noggin DMEM or RPMI for approximately 24 hours (day 7) to 48 hours (day 8).
Specifically, Applicants generate PDX1-positive foregut endoderm cells over a period of approximately 2
DMEM-high glucose has been used since 2003, and all patent and non-patent disclosures to that time use DMEM-high glucose even without any mention of "DMEM-high glucose" or the like. This is in part due to manufacturers such as Gibco not using their DMEM-high glucose.
M is not named as such, e.g., DMEM (Cat No. 11960) and Kno
It should be noted that, as of the filing date of this application, Gibco offers additional DMEM products, but has traditionally not labeled certain DMEM products containing high glucose, such as Knockout DMEM (Cat No. 10829-018). Thus, in each instance where DMEM is mentioned, it can be presumed that DMEM with high glucose is meant, which has been obvious to others conducting research and development in this field. Further details describing the use of exogenous high glucose are provided in Example 21. Additionally, ROCK inhibitors or rho kinase inhibitors, such as Y-27632, can be used to enhance growth, survival, proliferation, and promote cell-cell adhesion. PDX1-positive foregut cells generated at stage 3 were found to be able to express PDX1-positive foregut cells.
DX1 and HNF6, as well as SOX9 and PROX, were co-expressed at stage 1
and markers indicative of definitive endoderm cells or foregut endoderm cells described in stage 2 (PDX1-negative foregut endoderm) cells or PDX1-positive foregut endoderm cells are not appreciably co-expressed.

The Stage 3 method above is one of four stages for generating PECs. In addition to Noggin, KAAD-cyclopamine and retinoids, to generate endocrine precursors/progenitors and endocrine cells, as described in detail in Examples 9-24 below,
Activin, Wnt and Heregulin can be used alone and/or in combination to inhibit N
Repressing GN3 expression while maintaining good cell clumps, see Examples 8, 9 and 10.

For stage 4 (days 8-14) PEC generation, medium was taken from stage 3 and
The medium is replaced with DMEM containing 1% vol/vol B27 supplement plus 50 ng/mL KGF and 50 ng/mL EGF, and sometimes further 50 ng/mL Noggin and ROCK inhibitor, and further containing Activin alone or in combination with Heregulin. These new methods result in pancreatic progenitor cells that co-express at least PDX1 and NKX6.1 and PTF1A. These cells do not appreciably express markers indicative of definitive endoderm or foregut endoderm (PDX1-negative foregut endoderm) cells as described above in stages 1, 2, and 3. See FIG. 44.

Alternatively, cells from stage 4 were cultured in DMEM containing 1% v
The endocrine precursors/precursors generated at stage 5 can be further differentiated in medium with 100 mol/vol B27 supplement for about 1-6 days (preferably about 2 days, i.e., days 13-15) to generate endocrine precursor/precursor or precursor type cells and/or monohormonal and polyhormonal pancreatic endocrine cells.
They co-express at least CHGA, NGN3 and Nkx2.2, and do not appreciably express markers indicative of definitive endoderm or foregut endoderm (PDX1-negative foregut endoderm) as described above for stages 1, 2, 3 and 4 for PEC generation. See FIG.

For PEC generation, the PDX1-positive pancreatic endoderm generated at stage 4 is loaded into a macroencapsulation device, fully contained, and implanted into a patient, and the PDX1-positive pancreatic endoderm cells are allowed to mature in vivo into pancreatic hormone-secreting cells, or pancreatic islets, e.g., insulin-secreting beta cells (also referred to as "in vivo function"). Encapsulation of PDX1-positive pancreatic endoderm cells and in vivo insulin production are described in U.S. patent application Ser. No. 12/61, filed Dec. 13, 2003.
No. 8,659 (the '659 application), entitled ENCAPSULATION OF PANCRE
ATIC LINEAGE CELLS DELIVERED FROM HUMAN PL
The '659 application is a joint application of Provisional Patent Application No. 61/114,857, entitled ENCAPS
ULATION OF PANCREATIC PROGENITORS DELIVERY
D FROM HES CELLS, filed Nov. 14, 2008; and U.S. Provisional Patent Application No. 61/121,084, entitled ENCAPSULATION OF PANCREAS
TIC ENDODERM CELLS, filed December 9, 2008; now claims priority to U.S. Pat. Nos. 8,278,106 and 8,424,928.
The methods, compositions, and devices described herein presently represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art that are encompassed within the spirit of the invention and are defined by the scope of the disclosure. Thus, it will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

For example, activin A, a member of the TGFβ superfamily of growth factors or signaling proteins, is used to generate definitive endoderm from pluripotent cells, such as hES cells and iPS cells, although other TGFβ superfamily members, such as GDF-8 and GDF-11, can be used in the preparation of pluripotent cells, see International Patent Application No. PCT/US2008/065686.
No., Title: GROWTH FACTORS FOR PRODUCTION OF DEF
The cells can be used to generate definitive endoderm, such as that described in INITIAL ENDODERM, filed June 3, 2008.

In different situations, activin alone or in combination with Wnt and heregulin can suppress and inhibit the expression of NGN3 at high and low levels; at low levels, alone or in combination with heregulin, or at high levels, Wnt can suppress and inhibit the expression of NGN3.
In combination with T and heregulin, cell mass and therefore yield can be maintained.
See Examples 8, 9 and 10.

For PEC generation, stage 2 PDX1-negative foregut endoderm cells were cultured with stage 3 PDX1-negative foregut endoderm cells.
Retinoic acid (RA) is used to differentiate 4-[(
Other retinoids or retinoic acid analogs such as 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (or TTNPB) and similar analogs (e.g., 4-HBTTNPB) can be used. For endocrine cell and endocrine precursor/precursor cell generation, either retinoic acid or its analogs can also be added during stage 6 and/or stage 7 to induce hormone gene expression. See Example 13 and Example 16.

Noggin is a protein that inactivates members of the TGFβ superfamily of signaling proteins, such as bone morphogenetic protein-4 (BMP4). However, other BMP4 inhibitors, such as chordin and Twisted Gastrulation (Tsg), or anti-BMP neutralizing antibodies, can also prevent the binding of BMPs to their cell surface receptors, thereby effectively inhibiting BMP signaling. Alternatively, the human Noggin gene has been cloned and sequenced. U.S. Pat. No. 6,023,436 (Patent Publication No. 2002) discloses that Noggin inhibits BMP signaling by inhibiting the binding of BMPs to their cell surface receptors.
Analysis of the Noggin sequence reveals a carboxy-terminal region with homology to Kunitz-type protease inhibitors, potentially indicating that it may be a potential target for other Kunitz-type protease inhibitors.
It has been shown that nitz-type protease inhibitors may have similar effects on inhibiting BMPs.Example 15 describes the use of noggin to increase the generation of the non-endocrine (CHGA-) subpopulation.

Finally, this specification and the '659 application; U.S. Design Application No. 29/447,944;
No. 29/408,366; No. 29/408,368; No. 29/423,365
No. 61/774,443, entitled SEMIPERMEABLE MACRO
IMPLANTABLE CELLULAR ENCAPSULATION DEVICE
The macroencapsulation devices described in CES, filed March 7, 2013, are again merely exemplary and are not intended to limit the scope of the invention, particularly with respect to the size of the device, the presence of multiple chambers or subcompartments within the device, or the presence of multiple ports,
Furthermore, variations in device design, such as device loading and extraction mechanisms, are all within the scope of the present invention. Thus, it will be apparent to those skilled in the art that various substitutions and modifications to the differentiation methods described herein, as well as to the containment devices, can be made to the invention disclosed herein without departing from the scope and spirit of the present invention. See Example 10, Example 12, and Example 18.

For some processes for differentiating pluripotent cells into definitive endoderm cells, the above-mentioned growth factors are provided to the cells such that the growth factors are present in the culture at a concentration sufficient to promote differentiation of at least a portion of the pluripotent cells into definitive endoderm cells and cells of the pancreatic lineage. In some processes, the above-mentioned growth factors are provided to the cells at a concentration sufficient to promote differentiation of at least a portion of the pluripotent cells into definitive endoderm cells and cells of the pancreatic lineage.
ng/mL, at least about 10 ng/mL, at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, at least about 100 ng/mL, at least about 200 ng/mL, at least about 300 ng/mL, at least about 400 ng/mL
L, at least about 500 ng/mL, at least about 1000 ng/mL, at least about 2
000 ng/mL, at least about 3000 ng/mL, at least about 4000 ng/mL
, at a concentration of at least about 5000 ng/mL or greater than about 5000 ng/mL. Still other agents or growth factors are present in the cell culture at a concentration of at least 0.1 mM or greater than 10 mM.
M or greater.

In certain processes, the pluripotent stem cell differentiation agent and/or growth factor are removed from the cell culture after addition. For example, they can be removed within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days after adding the growth factor. In a preferred process, they are removed about 4 days after adding the growth factor. Removal of the agent and/or growth factor can be achieved by changing the medium in the absence of the agent and/or growth factor, or by using another agent that inhibits the function of the agent and/or growth factor.

In a preferred embodiment, the stage 3 cell culture includes, but is not limited to, agents capable of blocking, suppressing, inhibiting, etc., cells committed to the endocrine lineage (CHGA+). Such agents include activin, heregulin and WNT and combinations of the three as well as agents that promote differentiation and/or inhibit non-endocrine multipotent pancreatic progenitor subpopulations (CHGA+).
induced expression of markers indicative of endocrine lineage commitment (CHGA-) and cells committed to the endocrine lineage (CHGA+)
Markers indicative of cells committed to the endocrine lineage (CHGA+) include, but are not limited to, NG
N3, NKX2.2 and others.

In another preferred embodiment, the stage 4 cell culture includes agents capable of preventing, suppressing, inhibiting, etc., cells committed to the endocrine lineage (CHGA+), including but not limited to activin and heregulin and combinations of the two, in an amount effective to promote differentiation and/or induce expression of markers indicative of non-endocrine multipotent pancreatic progenitor subpopulations (CHGA-) and prevent or minimize expression of markers of endocrine lineage committed cells (CHGA+). Markers indicative of endocrine lineage committed cells (CHGA+) include, but are not limited to, NGN3, NK
X2.2 and others. At least NGN3 at stage 3 and stage 4
Means for inhibiting the expression of β-glucosamine during the course of treatment are described in detail in Examples 8-21 below.

In one embodiment, the stage 4 cell culture is cultured at stage 5 to express at least noggin,
Further differentiation is performed using KGF, EGF, and Notch signaling or pathway inhibitors. Markers indicative of endocrine differentiation include, but are not limited to, NGN3, NK
X2.2 and others. In a preferred embodiment, agents are used in stages 1-7 that simulate or effectively mimic in vitro those observed in in vivo developmental studies. For example, stage 3 according to the present invention and Table 17
and stage 4, using agents that can delay, block, suppress and/or inhibit endocrine differentiation or agents that can delay, block, suppress and/or inhibit markers indicative of endocrine differentiation, including, but not limited to, NGN3 and NKX2.2. During stages 5, 6 and/or 7, the cell cultures are cultured with agents that can induce, increase and/or promote endocrine differentiation, for example, using agents that can induce, increase and/or promote endocrine differentiation, such as gamma-secretarase inhibitors.
In summary, cell culture conditions at stages 3 and 4 suppress the endocrine phenotype, while cell culture conditions from stages 5, 6, and/or 7 suppress endocrine phenotypes including, but not limited to, insulin (INS), glucagon (GCG), somatostatin (SST), pancreatic polypeptide (PP), ghrelin (GHRL), solute carrier family 30 member 8 (SLC30A), and thymocyte endothelial cell line (TCL) phenotype.
8), glucose-6-phosphatase 2 (G6PC2), prohormone convertase 1 (P
The endocrine phenotype, including CSK1) and glucose kinase (GCK), is progressively induced.

In one embodiment, PDX1-positive pancreatic endoderm cells are treated with a Notch signaling inhibitor, e.g., R0492, used alone or in combination with a retinoid, such as retinoic acid.
PDX1-positive pancreatic endoderm cells are differentiated into endocrine cells and endocrine precursor/progenitor cells by continued incubation in the presence of a gamma sequestrantase inhibitor, such as 9097.
The presence of the Notch signaling inhibitor induces the expression of NGN3 during stage 5. In other embodiments, the gamma selectase inhibitor is provided at the beginning of the differentiation process, for example at the pluripotent stage, and remains in the cell culture throughout the differentiation into pancreatic islet hormone-expressing cells. In yet other embodiments, the gamma selectase inhibitor is added after differentiation has begun, but before differentiation into the PDX1-positive foregut endoderm stage. In preferred embodiments, the gamma selectase inhibitor is provided to the cell culture or cell population at about the same time that the differentiation factor that promotes the conversion of definitive endoderm to PDX1-positive endoderm is provided. In other preferred embodiments, the gamma selectase inhibitor is provided to the cell culture or cell population after a substantial portion of the cells in the cell culture or cell population have differentiated into PDX1-positive foregut endoderm cells.

In another embodiment, cell cultures from stage 5 are further differentiated at stages 6 and 7. During these stages, agents are used that can properly specify endocrine precursor/precursor or progenitor cells for in vivo differentiation into more developmentally advanced and/or mature endocrine cells. Such agents include:
These include, but are not limited to, retinoids or retinoic acid, BMPs, nicotinamide,
IGF, hedgehog protein, etc. In a preferred embodiment, TTNPB
(4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid or arotinoid acid) is a selective and highly potent retinoic acid analog with affinity for the nuclear transcription factors retinoic acid receptors (RAR) alpha, beta, and gamma. TTNPB and other retinoic acids or retinoic acid analogs result in ligand-activated transcription of genes that have retinoic acid response elements. Thus, other agents or ligands that can activate retinoic acid response elements or bind to any of the RARs are potentially useful and amenable to the present invention to promote endocrine cell differentiation.

In another embodiment, cell cultures from stage 5 or stage 6 or stage 7 can be further treated with matrigel alone or in combination with a rho kinase inhibitor to improve cell attachment. Alternatively, a rho kinase inhibitor can be added at any stage, stage 5, stage 6, or stage 7, at a sufficient concentration to promote cell survival, cell mass, and yield. Alternatively, cell cultures can be dissociated and reassociated between stages 6 and 7 or between stages 6 and 7 to produce non-endocrine multipotent pancreatic progenitor subpopulations, including but not limited to, CHGA, ...
-) can be used to remove or deplete unwanted cell types.
Please refer to 4.

In yet another embodiment, any of stages 1-7 is administered on the 1st, 2nd, 3rd, 4th, 5th, or 6th day of treatment.
You can extend your stay for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days.
It may be shortened by 3, 4, 5, 6, 7 or more days. For example, according to one preferred embodiment and Table 17, Stage 6 occurs over about 6 days and Stage 7 occurs over about 10 days.
The first stage occurs over a period of approximately nine days. However, both of these stages can be shortened to accommodate the generation of endocrine precursors/precursors, e.g., days 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81
It can be extended to 6, 37, 38, 39, 40 or more days.

In another embodiment, a method is provided for generating more developmentally advanced PECs or pancreatic endocrine precursors/progenitors or pancreatic endocrine cells. In one aspect of the invention, single pancreatic hormone expressing endocrine cells, such as I, that are co-positive for PDX1 and NKX6.1, are generated.
Immature beta cells are provided that co-express NS, PDX1 and NKX6.1.
In another embodiment, the developmentally advanced cells may be PEC cultures that consist primarily of non-endocrine pancreatic progenitor cells expressing at least PDX and NKX6.1, but not or minimally of endocrine lineage-committed cells (CHGA+) expressing at least CHGA+, NGN3 and/or NKX2.2. These PEC cultures are believed to be more similar to those seen in vivo in developmental studies of pancreatic differentiation.
Developmentally advanced. Jorgensen et al. (2007), An Illustrated
ed Review of Early Pancreas Development
in the Mouse, Endocrine Reviews 28(6):685
-705 and Rukstalis and Habener (2009), Neurology
nin3: A master regulator of panic is
Let differentiation and regeneration, Isl
See, e.g., J. Immunol. 1(3):177-184. Another example of a developmentally advanced cell or cell culture is a reaggregated cell culture that has been detached and reaggregated or reassociated, and thus consists of a more homogenous population of cells. This more homogenous population of cells, in one aspect of the invention, includes cells or subpopulations from stage 3 and stage 4 that are co-positive for PDX1 and NKX6.1, or, but is not limited to, non-endocrine multipotent pancreatic progenitor (CHGA-) cells, or residual/triple negative cells (CHGA-/PDX1
-/NKX6.1-), or do not contain earlier stage cell types, e.g., pancreatic endocrine precursors/precursors or endocrine cells, that are primarily composed of single hormone expressing cells, including multi-hormone expressing cells (i.e., cells that express more than one hormone in any one cell type).

In another embodiment, the cell cultures provided in the present invention are preferably properly specialized, which has a specific and certain meaning in the field of developmental biology. In terms of developmental biology, it is the mechanism by which cells acquire or specify or properly specify distinct fates. Since differentiation of cell cultures in vitro lacks the temporal organization and cues typical of in vivo developmental studies, the use and reliability of cell markers (surface markers or internal markers such as transcription factors), in particular signatures or prominent cell markers that indicate a particular cell type, is essential. Thus, reference to properly specialized cells, cell cultures, subpopulations and populations means that the cells have distinct fates, and that their fates are more certain and determined based on the expression of their markers, the profile of markers they express, and, importantly, the markers they do not express. Proper specification of cell cultures in vitro allows for the differentiation of similar cells in vivo.
Thus, in a preferred embodiment, the in vivo developmental test is
A guide to in vitro differentiation and characterization of properly specialized cells.

In some embodiments of the invention described herein, exendin 4 is provided to the differentiating cell culture or cell population at about the same time as the gamma selenase inhibitor. In certain embodiments, exendin 4 is provided to the cell culture or cell population at a concentration of at least about 0.1
It is provided to be present at concentrations from ng/mL to 1000 ng/mL.

It will be appreciated that expression of the NGN3, NKX2.2 and/or PAX4 markers is induced in endocrine precursor/progenitor cells across a variety of different levels depending on the state of differentiation. As such, in some embodiments described herein, expression of the NGN3, NKX2.2 and/or PAX4 markers in endocrine precursor/progenitor cells or cell populations is induced across a variety of different levels depending on the state of differentiation.
Non-endocrine precursor/progenitor cells or cell populations, e.g., pluripotent stem cells, definitive endoderm cells, PD
NGN3, NKX2.2 in X1-positive foregut endoderm cells, immature pancreatic islet hormone-expressing cells, mature pancreatic islet hormone-expressing cells, extraembryonic endoderm cells, mesoderm cells and/or ectoderm cells
and/or at least about 2-fold to at least about 10,000-fold increase in expression of the PAX4 marker
In another embodiment, the expression of NGN3 in an endocrine precursor/progenitor cell or cell population is
Expression of the NKX2.2 and/or PAX4 markers is at least about 4-fold, at least about 6-fold to 10,000-fold greater than expression of the NGN3, NKX2.2 and/or PAX4 markers in non-endocrine precursor/progenitor cells or cell populations, e.g., pluripotent stem cells, definitive endoderm cells, PDX1 positive foregut endoderm cells, immature pancreatic islet hormone expressing cells, mature pancreatic islet hormone expressing cells, extraembryonic endoderm cells, mesoderm cells and/or ectoderm cells. In some embodiments, expression of the NGN3, NKX2.2 and/or PAX4 markers in endocrine precursor/progenitor cells or cell populations is at least about 4-fold, at least about 6-fold to 10,000-fold greater than expression of the NGN3, NKX2.2 and/or PAX4 markers in non-endocrine precursor/progenitor cells or cell populations, e.g., pluripotent stem cells, definitive endoderm cells, PDX1 positive foregut endoderm cells, immature pancreatic islet hormone expressing cells, mature pancreatic islet hormone expressing cells, extraembryonic endoderm cells, mesoderm cells and/or ectoderm cells.
Alternatively, expression of the PAX4 marker may be indicative of NGN3, NKX2.2 and/or PAX4 in non-endocrine precursor/progenitor cells or cell populations, such as pluripotent cell-like iPS cells and hES cells, definitive endoderm cells, PDX1 positive foregut endoderm cells, immature pancreatic islet hormone expressing cells, mature pancreatic islet hormone expressing cells, extraembryonic endoderm cells, mesoderm cells and/or ectoderm cells.
Much higher than the expression of the marker.

Another embodiment of the invention is a composition, such as a cell culture or cell population, comprising human cells, including human endocrine precursor/progenitor cells, wherein at least about 2% of the human cells express NGN3.
Marker expression was AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP,
In another embodiment, the composition comprises at least about 5% to 98% of human cells expressing greater than or equal to the expression of the markers CHGA, INS, GCG, SST, GHRL, and/or PAX6.
In the study, the expression of NGN3 markers was significantly higher than that of AFP, SOX7, SOX1, ZIC1, NFM, and M
AFA, SYP, CHGA, INS, GCG, SST, GHRL, and/or PAX
In some embodiments, the expression of the following markers in the cell culture or cell population is greater than 6:
The expression of NGN3 was associated with AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP,
The percentage of human cells with greater than expression of CHGA, INS, GCG, SST, GHRL, and/or PAX6 markers is calculated without considering feeder cells.

Some embodiments of the invention provide a composition, such as a cell culture or cell population, comprising human endocrine precursor/progenitor cells, wherein expression of NKX2.2 and/or PAX4 is increased in at least about 2% to at least about 98% of the human cells, and expression of AFP, SOX7, SOX1, ZI
It will be appreciated that the present invention relates to compositions that have greater than expression of markers NKX2.Cl, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL, and/or PAX6. In some embodiments, the compositions have greater than expression of markers NKX2.Cl in at least about 5% of human cells to 98% of human cells.
2 and/or PAX4 expression is AFP, SOX7, SOX1, ZIC1, NFM, M
AFA, SYP, CHGA, INS, GCG, SST, GHRL, and/or PAX
In some embodiments, the expression of the following markers in the cell culture or cell population is greater than 6:
NKX2.2 and/or PAX4 expression is associated with AFP, SOX7, SOX1, ZIC1,
The percentage of human cells with greater than expression of the NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL, and/or PAX6 markers is calculated without considering feeder cells.

The processes described herein can be used to produce endocrine precursor/cells that are substantially free of other cell types.
In some embodiments of the invention, the endocrine precursor/progenitor cell populations or cell cultures produced by the methods described herein can be AFP,
In some embodiments, the endocrine precursor/progenitor cell population of the cell culture generated by the methods described herein is substantially free of cells that significantly express the markers AFP, SOX7, SOX1, ZIC1, NFM, M
AFA, SYP, CHGA, INS, GCG, SST, GHRL, and/or PAX
The cells are substantially free of cells that significantly express the six markers.

Yet another process for generating immature pancreatic islet hormone-expressing cells disclosed herein provides HGF to the cells, thus providing HGF to the cell culture or cell population.
In some embodiments, HGF is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor/progenitor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, HGF is present in the cell culture or cell population at a concentration of at least about 1 ng/mL, at least about 5 ng/mL to 10
It is present at a concentration of 0.00 ng/mL.

Yet another process for generating immature pancreatic islet hormone-expressing cells disclosed herein provides IGF1 to the cells, thus providing in the cell culture or cell population:
IGF1 is present in the cell culture or cell population at a concentration of at least about 1 ng/mL to 1000 ng/mL, such that IGF1 is present in a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor/precursor cells into immature pancreatic islet hormone-expressing cells.

In certain embodiments of the processes for generating immature pancreatic islet hormone-expressing cells described herein, one or more of nicotinamide, exendin 4, HGF, and IGF1 are provided after the previously provided one or more differentiation factors are removed from the cell culture. In other embodiments, nicotinamide, exendin 4, HGF, and IGF1 are provided after the previously provided one or more differentiation factors are removed from the cell culture.
One or more of nicotinamide, exendin 4, HGF, and IGF1 are provided to a cell culture or cell population that includes one or more differentiation factors provided prior to or at about the same time as one or more of nicotinamide, exendin 4, HGF, and IGF1. In a preferred embodiment, the differentiation factors provided prior to or at about the same time as one or more of nicotinamide, exendin 4, HGF, and IGF1 include, but are not limited to, DAPT, FGF-10, KAAD-cyclopamine, activin A, activin B, BMP4, and/or RA.

Another embodiment of the invention is a composition, such as a cell culture or cell population, comprising human cells, including human immature pancreatic islet hormone-expressing cells, wherein at least about 2% of the human cells contain M
AFB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, PDX
1, HB9, GHRL, IAPP, INS, GCG, SST, PP, and/or C-
Expression of peptide markers: NGN3, MAFA, MOX1, CER, POU5F1, AF
In other embodiments, the expression of MAFB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, P, SOX7, SOX1, ZIC1 and/or NFM markers is greater than or equal to 10% of the human cells, at least about 10% to 95% of the human cells, or at least about 98% of the human cells.
Expression of PDX1, HB9, GHRL, IAPP, INS, GCG, SST, PP, and/or C-peptide markers is
, greater than expression of AFP, SOX7, SOX1, ZIC1 and/or NFM markers.

In certain embodiments of the invention, the cell cultures and/or cell populations comprising immature pancreatic islet hormone-expressing cells also comprise medium comprising one or more secreted hormones selected from ghrelin, insulin, somatostatin and/or glucagon. In other embodiments, the medium comprises C-peptide. In preferred embodiments, the concentration of the one or more secreted hormones or C-peptide in the medium ranges from at least about 1 pmol of ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of intracellular DNA to at least about 1000 picomoles (pmol) of ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of intracellular DNA.

Methods for Producing Insulin In Vivo In some embodiments, the in vitro derived pancreatic progenitor cells or PDX described above are
The -1 positive pancreatic endoderm cells or their equivalent are then transplanted into a mammalian subject. These methods are described in International Application No. PCT/US2007/015536, entitled "METHODS OF TRANSPLANTATION
RODUCING PANCREATIC HORMONES” and Kroon et al.
2008) supra. In a preferred embodiment, the mammalian subject is a human subject. Particularly preferred subjects are those identified as having a condition that limits the subject's ability to produce sufficient levels of insulin in response to physiologically high blood glucose concentrations. The range of blood glucose levels that constitute physiologically high blood glucose levels for any particular mammalian species can be readily determined by one of skill in the art. Any sustained blood glucose level that results in a recognized disease or condition is considered a physiologically high blood glucose level.

Further embodiments of the present invention include the production of in vitro pluripotent stem cells or their progeny, such as PDX-1 positive foregut endoderm cells, PDX-1 positive pancreatic endoderm or pancreatic progenitor cells, N
The present invention relates to in vivo insulin-secreting cells derived from multipotent cells, such as endocrine precursors/precursors, such as GN3-positive endocrine precursors/precursors, or functional differentiated hormone-secreting cells, such as insulin-secreting cells, glucagon-secreting cells, somatostatin-secreting cells, ghrelin-secreting cells, or pancreatic polypeptide-secreting cells. Any of the above terminally differentiated cells or multipotent cells can be transplanted into a host, or a mammal, and matured into a physiologically functional hormone-secreting cell, such as a cell that secretes insulin in response to the host blood glucose level. In a preferred embodiment, the cells do not form teratomas in vivo, and if so formed, they remain localized in the area of transplantation and can be easily excised or removed. In a particularly preferred embodiment, the cells do not contain any karyotypic abnormalities during the differentiation process in vitro, or when transplanted in vivo into a mammal, or when maturing and developing into functional islets in vivo.

Additionally, embodiments described herein relate to engineered or genetically modified pluripotent cells, multipotent cells or differentiated cells derived based on the description provided herein from pluripotent cells, such as human iPS cells, where the iPS cells are derived from hE
Having demonstrated similar physiological functions and gene marker expression profiles as S cells and hES-derived cells, iPS cells are predicted to have similar physiological properties in vivo.

Methods of Encapsulating Pancreatic Progenitors In some embodiments, the pluripotent, multipotent and differentiated cell compositions described herein can be encapsulated in biological and/or non-biological mechanical devices, where the encapsulated device separates and/or segregates the cell composition from the host.

The method of encapsulation is described in U.S. Patent Application 61/114,857, filed November 14, 2008;
Title: ENCAPSULATION OF PANCREATIC PROGENITOR
S DERIEVED FROM HES CELLS, and U.S. Patent Application No. 61/12
No. 1,086, filed December 12, 2008, entitled ENCAPSULATION OF
This is described in detail in PANCREATIC ENDODERM CELLS.

In one embodiment, the encapsulation device contains pluripotent derived cells, e.g., endocrine or endocrine precursors/precursors such as PDX-1 positive foregut endoderm cells, PDX-1 positive pancreatic endoderm or progenitor cells, NGN3 positive endocrine precursors/precursors, or functional differentiated hormone secreting cells such as insulin-secreting cells, glucagon-secreting cells, somatostatin-secreting cells, ghrelin-secreting cells, or pancreatic polypeptide-secreting cells, within a semi-permeable membrane that prevents the passage of the transplanted cell population and retains them within the device while allowing the passage of certain secreted polypeptides, e.g., insulin, glucagon, somatostatin, ghrelin, pancreatic polypeptide, etc. Alternatively, the device has multiple membranes, including a vascularized membrane.

Use of agents to enhance and promote the growth, survival, proliferation and cell-cell adhesion of human pluripotent stem cells, such as hES and iPS cells The transmission of extracellular signals across membranes can affect cell regulation, which in turn can modulate biochemical pathways within the cell. Protein phosphorylation represents one way in which intracellular signals are propagated from molecule to molecule, ultimately resulting in a cellular response. These signal transduction cascades are highly regulated and often overlapping, as evidenced by the presence of many protein kinases and phosphatases. In humans, protein tyrosine kinases have been reported to have been found to play an important role in the development of many pathologies, including diabetes, cancer, and have also been linked to a wide variety of congenital syndromes. Serine-threonine kinases, such as Rho kinase, are a class of enzymes that, when inhibited, may be relevant for the treatment of human diseases, including diabetes, cancer, and various inflammatory cardiovascular disorders and AIDS. The majority of inhibitors identified to date act at the ATP binding site.
Such ATP-competitive inhibitors have demonstrated selectivity due to their ability to target less conserved regions of the ATP-binding site.

The Rho kinase family of small GTP-binding proteins consists of at least 10 kinases, including Rho A-E and RhoG, Rac1 and Rac2, Cdc42, and TC10.
The inhibitors are often referred to as ROK inhibitors or ROCK inhibitors or Rho kinase inhibitors, which are used interchangeably herein.
The effector domains of hoA, RhoB, and RhoC have the same amino acid sequence,
Rho kinase acts as the first downstream mediator of Rho and exists as two isoforms: α (ROCK2) and β (ROCK1). Rho kinase family proteins have a catalytic (kinase) domain in their N-terminal domain, a coiled-coil domain in their central part, and a putative pleckstrin homology (PH) domain in their C-terminal region. Rho kinase is a kinase that acts as a first downstream mediator of Rho and exists as two isoforms: α (ROCK2) and β (ROCK1). Rho kinase family proteins have a catalytic (kinase) domain in their N-terminal domain, a coiled-coil domain in their central part, and a putative pleckstrin homology (PH) domain in their C-terminal region.
The Rho-binding domain is located in the C-terminal portion of the coiled-coil domain, and binding to the GTP-bound form of Rho leads to enhanced kinase activity. The Rho/Rho kinase-mediated pathway is involved in the binding of angiotensin II, serotonin, thrombin, endothelin-1,
Rho kinase plays an important role in the signal transduction initiated by many agonists, including norepinephrine, platelet-derived growth factor, ATP/ADP and extracellular nucleotides, and urotensin II. Through the modulation of its target effector/substrate, Rho kinase plays an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, cell adhesion and motility, and gene expression. Due to the role that Rho kinase protein plays in mediating several cellular functions that are recognized to be related to the pathogenesis of arteriosclerosis, inhibitors of these kinases may also be useful for treating or preventing various arteriosclerotic cardiovascular diseases, and are involved in endothelial contraction.

In some embodiments, including but not limited to, the Rho kinase inhibitor Y-2763
2. Fasudil (also called HA1077), H-1152P and ITS (insulin/transferrin/selenium; Gibco), Wf-536, Y-30141 (described in U.S. Pat. No. 5,478,838) and their derivatives, and ROCK
Agents that promote and/or support cell growth, survival, proliferation, and cell-cell adhesion, including antisense nucleic acids against, RNA interference-inducing nucleic acids (e.g., siRNA), competitive peptides, antagonist peptides, inhibitory antibodies, antibody-scFV fragments, dominant negative variants, and expression vectors thereof, are added to various cell culture medium conditions. In addition, other small molecule compounds are known as ROCK inhibitors, and such compounds or derivatives thereof can also be used in the present invention (see, for example, U.S. Patent Application No. 20050022144).
No. 209261, No. 20050192304, No. 20040014755, No. 20040002508, No. 20040002507, No. 2003012534
4 and 20030087919, and International Patent Publication No. 2003/06222.
7, 2003/059913, 2003/062225, 2002/
(see US Pat. Nos. 076976 and 2004/039796).

Combinations of one or two or more ROCK inhibitors can also be used in the present invention. These agents function, in part, by promoting the reassociation of dissociated hES cells, iPS or differentiated cell cultures, such as definitive endoderm, foregut endoderm, pancreatic endoderm, pancreatic epithelium, pancreatic progenitor populations, endocrine progenitors and populations. Similarly,
The agents may also function when cell dissociation is not performed. Increased growth, survival, proliferation and cell-cell adhesion of human pluripotent stem cells has been demonstrated whether the cells are generated from cell aggregates in suspension or in adherent plate cultures (with or without components of the extracellular matrix, with or without serum, with or without fibroblast feeders, with FGF
This was achieved independently of whether they were generated from IL-1 or IL-2 (with or without activin, with or without activin). The increased survival of these cell populations facilitates and improves the purification system using cell sorters, thus allowing for improved recovery of cells.
The use of Rho kinase inhibitors such as Y27632 may also allow the expansion of differentiated cell types by promoting their survival during stepwise passaging of dissociated single cells or from cryopreservation. Rho kinase inhibitors such as Y27632 have been tested on human pluripotent stem cells (e.g., hES cells and iPS cells) and their differentiated cells, but R
Ho kinase inhibitors are also useful in the treatment of other cell types, such as, but not limited to, intestine,
It can be applied to epithelial cells including lung, thymus, kidney and nervous system cell types as well as retinal pigment epithelium.

The concentration of the ROCK inhibitor in the cell culture medium is such that the desired effect is achieved.
For example, as long as the enhancement, increase, and/or promotion of cell growth, survival, proliferation, and cell-cell adhesion is realized, the present invention is not limited to those described in the following examples.
It will be appreciated by those skilled in the art that inhibitors may need to be optimized under different conditions. For example, when using Y-27632, the preferred concentration is about 0.01 to about 1000.
μM, more preferably from about 0.1 to about 100 μM, and even more preferably from about 1.0 to about 50 μM.
M, most preferably about 5-20 μM. When fasudil/HA1077 is used, it can be used at about 2 or 3 times the Y-27632 concentration mentioned above.
When using Y-27632, the amount is approximately a fraction of the amount of the Y-27632 concentration mentioned above, e.g., about 1/1
It can be used at 0, 1/20, 1/30, 1/40, 1/50 or 1/60.
The concentration of ROCK inhibitor used will depend, in part, on the biological activity and potency of the inhibitor and the conditions under which it is used.

The time and duration of treatment with a ROCK inhibitor may or may not be limited depending on the desired effect, such as enhancing, increasing, and/or promoting growth, survival, proliferation (cell population) and cell-cell adhesion. However, the addition of a ROCK inhibitor can also affect differentiation in a surprising manner, as better described in Example 7. The following examples include a 12-well platelet count of approximately 12% of cells.
Human pluripotent stem cell cultures and/or differentiated cell cultures that have been treated for 1 hour, 24 hours, 48 hours, or more are described.

The density of human pluripotent stem cell cultures treated with ROCK inhibitors also increased, as
There is no limit to the density as long as the desired effects, such as enhanced, increased, and/or promoted cell growth, survival, proliferation, and cell-cell adhesion, are achieved. The cell density of the seeded cells can be adjusted depending on a variety of factors, including, but not limited to, the use of adherent or suspension cultures, the particular recipe of the cell culture medium used, the growth conditions, and the intended use of the cultured cells. Examples of cell culture densities include, but are not limited to, 1000 cells/ml
0.01 x ¹⁰ cells per ml, 0.05 x ¹⁰ cells per ml, 0.1 x ¹⁰ cells per ml, 0.5 x ¹⁰ cells per ml, 1.0 x 10 cells per ml
0 ⁵ cells per ml, 1.2 x 10 ⁵ cells per ml, 1.4 x 10 ⁵ cells per ml,
1.6 x ¹⁰ cells per liter, 1.8 x ¹⁰ cells per ml, 2.0 x ¹⁰ cells per ^ml , 3.0 x ¹⁰ cells per ml, 4.0 x 10 cells per ml
cells per ml, 5.0 x ¹⁰ cells per ml, 6.0 x ¹⁰ cells per ml, 7.0 x ¹⁰ cells per ml, 8.0 x ¹⁰ cells per ml, 9.0 x 10 cells per ml.
0×10 ⁵ cells per ml, or 10.0×10 ⁵ cells per ml, or more, e.g.
Up to ^5x107 cells per mL, or more, or any value in between, were cultured with good cell growth, survival, proliferation and cell-cell adhesion.

Uses of Agents That Activate TGFβ Receptor Family Members In yet another embodiment, agents that activate TGFβ receptor family members include members of the TGFβ superfamily of growth factors, as described herein. As used herein, "TGFβ superfamily members" or equivalents include TGFβ1, TGFβ2, TF-β3, GDF-15, GDF-9, BMP-1,
5. BMP-16, BMP-3, GDF-10, BMP-9, BMP-10, GDF-6
, GDF-5, GDF-7, BMP-5, BMP-6, BMP-7, BMP-8, BMP
-2, BMP-4, GDF-3, GDF-1, GDF11, GDF8, activin βC,
βE, βA and βB, BMP-14, GDF-14, MIS, inhibin alpha, L
Refers to over 30 structurally related proteins, including subfamilies including efty1, Lefty2, GDNF, neurturin, persephin and artemin.
See Hang et al. (2002) Endocrine Rev. 23(6):787-823.

TGFβ family members can be replaced by or used in conjunction with TGFβ signaling pathway activators, which are compounds that stimulate one or more of the polypeptides or interactions involved in transducing or otherwise effecting changes in cellular properties in response to TGFβ family members. The TGFβ signaling pathway includes the TGFβ family members themselves. TGFβ superfamily members include:
They transmit signals to the nucleus by signaling through type II and type I serine-threonine kinase receptors and intracellular effectors known as Smads. These receptors fall into two subfamilies known as type I and type II receptors that act cooperatively to bind ligands and transmit signals (Attisano et al., Mol Cell 1999, 144:131-132).
Biol 16(3), 1066-1073 (1996)). Ligand binds to the cell surface of type I and II receptors and promotes activation of type I receptors by phosphorylation. This activated complex in turn activates intracellular Smads, which assemble into multisubunit complexes that regulate transcription. Members of the TGF-beta superfamily are divided into two signaling subgroups: those functionally related to TGF-β/activin, and those functionally related to BMP/G
Most TGFβ ligands are thought to first bind to the type II receptor, and then this ligand/type II receptor complex recruits or phosphorylates the type I receptor (Mathews, L S, Endocr Rev
15:310-325 (1994); Massague, Nature Rev: Mo
l Cell Biol. 1, 169-178 (2000)). Type II receptor kinases phosphorylate and activate type I receptor kinases, which then phosphorylate and activate Smad proteins. TGFβ/activin ligands bind to TGFβ and activin type II receptors, and through this activin-type pathway, they phosphorylate and activate Smad-2, Smad-
3, can activate Nodal and Lefty signals. BMP/GDF ligands bind to BMP type II receptors and can activate Smad1, Smad5, and Smad8. Derynck, R. et al., Cell 95, 737-740 (1
Upon ligand stimulation, Smads translocate into the nucleus and function as components of transcription complexes.

TGFβ signaling is positively and negatively regulated through various mechanisms. In positive regulation, the signal is amplified to a sufficient level for biological activity. TGFβ superfamily ligands bind to type II receptors, which recruit and phosphorylate type I receptors. Type I receptors then mediate the expression of receptor-regulated SMADs (R-SMADs,
R-SMAD/coSMAD complexes accumulate in the nucleus where they function as transcription factors and are involved in the regulation of target gene expression. For example, growth and differentiation factor 1, 2, 3, 5, and 6 are phosphorylated and can bind to common mediator Smads or co-SMADs.
, 6, 7, 8, 9, 10, 11, and 15. Also in a preferred embodiment, G
DF8 and GDF11 are TGFβ family members as well as TGFβ signaling pathway activators (positive regulators) that act by binding to the extracellular ligand-binding domain portion of the ActRII receptor and then forming a complex with ActRI, thereby leading to inhibition of the Smad7 negative regulator and phosphorylation of the Smad2/Smad3 complex, which associates with Smad4 to regulate the expression of specific genes.

As with the use of any agent, the concentration of any TGFβ superfamily member in the cell culture medium is not limited to those described in the Examples below, so long as the concentration is capable of achieving the desired effect, such as activating a TGFβ receptor family member. For example, when using activins, such as activin A and/or activin B, or GDF8 and GDF-11, preferred concentrations are about 1:1 or about 1:1.
0 to about 300 nM, more preferably about 50 to about 200 nM, even more preferably about 75 to about
The concentration can range from about 150 nM, and most preferably from about 100 to 125 nM. One of skill in the art can readily test any concentration and use standard techniques in the art to determine the effectiveness of such concentrations, for example, to assess differentiation by determining the expression and non-expression of a panel of genetic markers for any cell type.

Use of Agents to Generate Endocrine Cells In one embodiment, the present invention provides a method of generating pancreatic endoderm lineage type populations and/or subpopulations, particularly PECs and/or pancreatic endocrine lineage cells, using Notch pathway inhibitors, including but not limited to gamma selectase inhibitors, in an amount effective to promote endocrine differentiation and/or induce expression of certain markers that are characteristic of and indicative of endocrine differentiation. A number of gamma selectase inhibitors have been described.
See, e.g., U.S. Patent Nos. 6,756,511; 6,890,956; and 6,984
,626; 7,049,296; 7,101,895; 7,138,
See, e.g., Calbiochem (La Jolla, Calif.);
lif): Gamma Sectorase Inhibitor I, (GSI I), Z
-Leu-Leu-norleucine-CHO; gamma-secretaase inhibitor II; gamma-secretaase inhibitor III, (GSI III), N-benzyloxycarbonyl-Leu-
Leucinal; gamma-secretase inhibitor IV, (GSI IV), N-(2-naphthoyl)-Val-phenylalaninal; gamma-secretase inhibitor V, (GSI V),
N-benzyloxycarbonyl-Leu-phenylalaninal; gamma-secretase inhibitor VI, (GSI VI), 1-(S)-endo-N-(1,3,3)-trimethylbicyclo[2.2.1]hept-2-yl)-4-fluorophenylsulfonamide; gamma-secretase inhibitor VII, (GSI VII), methyloxycarbonyl-LL-C
HO; gamma-secretases inhibitor IX, (GSI IX), (DAPT), N-[N-(
3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester; gamma sequestrantase inhibitor X, (GSI X), {1S-benzyl-4R-
[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester; Gamma-secretase inhibitor XI, (GSI XI), 7-amino-4-
Chloro-3-methoxyisocoumarin; Gamma Sectorase Inhibitor XII, (GSI XI
I), Z-Ile-Leu-CHO; gamma-secretaase inhibitor XIII, (GSI X
III), Z-Tyr-Ile-Leu-CHO; gamma-secretases inhibitor XIV, (
GSI XIV), Z-Cys(t-Bu)-Ile-Leu-CHO; gamma-secretase inhibitor XVI, (GSI XVI), N-[N-3,5-difluorophenacetyl]-L-alanyl-S-phenylglycine methyl ester; gamma-secretase inhibitor X
VII, (GSI XVII), WPE-III-31C); gamma-secretases inhibitors XIX, (GSI XIX), (2S,3R)-3-(3,4-difluorophenyl)-
2-(4-fluorophenyl)-4-hydroxy-N-((3S)-2-oxo-5-
Phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-
Butyramide; Gamma Sectorase Inhibitor XX, (GSI XX), (Dibenzazepine (
DBZ), (S,S)-2-[2-(3,5-difluorophenyl)acetylamino]
-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propionamide, gamma-secretase inhibitor XXI, (GSI XX
I), (S,S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N
-(1-methyl-2-oxo-5-phenyl-2-,3-dihydro-1H-benzo[e]
[1,4]diazepin-3-yl)-propionamide; gamma 40 selectase inhibitor I, N-trans-3,5-dimethoxycinnamoyl-Ile-leucinal; gamma 4
0. Sectorase inhibitor II, N-tert-butyloxycarbonyl-Gly-Val
-Valinal; and Isovaleryl-V-V-Sta-A-Sta-
_O.C.3 .

In one aspect, the gamma sequestrantase inhibitor is Z-Leu-Leu-norleucine-CH
O, N-benzyloxycarbonyl-Leu-Leucinal, N-(2-naphthoyl)-
Val-phenylalaninal, N-benzyloxycarbonyl-Leu-phenylalaninal, 1-(S)-endo-N-(1,3,3)trimethylbicyclo[2.2.1
]hept-2-yl)-4-fluorophenylsulfonamide, menthyloxycarbonyl-LL-CHO, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-
S-Phenylglycine t-butyl ester, {1S-benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-
2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester, 7
-amino-4-chloro-3-methoxyisocoumarin, Z-Ile-Leu-CHO, Z-
Tyr-Ile-Leu-CHO, Z-Cys(t-Bu)-Ile-Leu-CHO,
N-[N-3,5-difluorophenacetyl]-L-alanyl-S-phenylglycine methyl ester, (2S,3R)-3-(3,4-difluorophenyl)-2-(4-fluorophenyl)-4-hydroxy-N-((3S)-2-oxo-5-phenyl-2,
3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-butyramide,
(S,S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(1
-Methyl-2-oxo-5-phenyl-2-,3-dihydro-1H-benzo[e][1,
4]diazepin-3-yl)-propionamide, N-trans-3,5-dimethoxycinnamoyl-1-leucinal, N-tert-butyloxycarbonyl-Gly
In one aspect of the invention, the renal disease is selected from the group consisting of one or more of: Isovaleryl-V-Val-valinal, Isovaleryl-V-V-Sta-A-Sta-OCH ₃ , and the renal disease is glomerular or tubular renal disease.
A gamma-secretase inhibitor is used at 3 mM, preferably 0.5-1 mM.

In one embodiment, insulin growth factor (IGF) is part of a complex system that cells use to communicate with their physiological environment and therefore can be used to differentiate PSCs into PECs and/or cells of the pancreatic endocrine lineage. This complex system (often referred to as the IGF "axis") is a cellular signaling pathway that mediates the differentiation of PSCs into PECs and/or cells of the pancreatic endocrine lineage through the interaction of two cell surface receptors (IGF1R and IGF2R).
, two ligands (insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2)), and six high-affinity IGF-binding proteins (IGFBP-1 to IGF
The IGFs are known to bind to IGF1R, insulin receptor, IGF-2 receptor, insulin-related receptor and possibly other receptors. Thus, in one aspect of the invention, growth factors that bind to these receptors include, but are not limited to, IGF1 and IGF2, as any growth factor, agent or morphogen that binds to any or a combination of these receptors will potentially have the same physiological effects as described and predicted herein. In one aspect of the invention, growth factors that bind to any or all of stages 1-7 according to Tables 8 and 17 as described herein include, but are not limited to, IGF1 and IGF2.
IGF is used at about 5-200 ng/mL, 10-100 ng/mL, 10-75 ng/mL, preferably 10-50 ng/mL, preferably 20-50 ng/mL.

In one embodiment, platelet-derived growth factor (PDGF) is used to differentiate PSCs into PECs and/or cells committed to the endocrine lineage (CHGA+). PDGF is a major protein growth factor that has been extensively described as a potent mitogen for many types of cells. PDGF belongs to a family of dimeric isoforms of polypeptide chains, A, B, C, and D, that act through distinct tyrosine kinase receptors: PDGFR-α and PDGFR-β. In one aspect of the invention, the ligand is PDGF or a functional equivalent thereof, including but not limited to PDGFaa, PDGFab, or PDGFbb, which bind to two types of receptors. In one aspect of the invention, a PDGF ligand is administered at about 5-100 ng/m2 in any or all of stages 1-7 according to Tables 8 and 17, as described herein.
L, 10 to 75 ng/mL, 10 to 50 ng/mL, preferably 10 to 20 ng/mL, of PDGF is used.

In one embodiment, nicotinamide, also known as niacinamide and nicotinic acid amide, which is the amide of nicotinic acid (vitamin B3/niacin), is used to differentiate PSCs into PECs and/or pancreatic endocrine lineage cells. Nicotinic acid, also known as niacin, is converted to nicotinamide in vivo, and although the vitamin functions of niacin and nicotinamide are identical, nicotinamide does not have the same pharmacological and toxic effects as niacin. Thus, other B vitamins or vitamin B derivatives that function similarly to nicotinamide are incorporated into the present invention. In one aspect of the present invention, a method for differentiation of PSCs into PECs and/or pancreatic endocrine lineage cells, as described herein, is provided comprising administering to the patient a 5-vitamin B vitamin supplement at any or all of stages 1-7 according to Tables 8 and 17, ... as described herein.
100 ng/mL, 10-75 ng/mL, 10-50 ng/mL, preferably 10-2
0 ng/mL nicotinamide is used.

In another embodiment, the hedgehog family of proteins is combined with the PSC to PEC and/or
or for differentiation into pancreatic endocrine lineage cells. The Hedgehog family of vertebrate intercellular signaling molecules provided by the present invention includes, but is not limited to,
Desert hedgehog (Dhh), Sonic hedgehog (Shh), Indian hedgehog (Ihh)
h) and Moonrat hedgehog (Mhh)
In one aspect, the present invention comprises at least four members, including
In any or all of stages 1 to 7 according to Tables 8 and 17, about 5 to 200 n
g/mL, 10-200 ng/mL, 10-150 ng/mL, preferably 10-100
ng/mL of hedgehog protein is used.

Having generally described the invention, a further understanding can be obtained by reference to certain specific examples provided herein, which are intended to be illustrative and not limiting.

It should also be understood that the above relates to preferred embodiments of the present invention, and that numerous modifications can be made thereto without departing from the scope of the present invention. The present invention is further illustrated in the following examples, which should not be construed as limiting its scope in any way. On the contrary, it should be clearly understood that resort can be made to various other embodiments, modifications and equivalents thereof, which may occur to those skilled in the art after reading the description herein, without departing from the spirit of the present invention and/or the scope of the appended claims.

Example 1
Differentiation of Human iPS Cells to Pancreatic Progenitors and Endocrine Cells via Definitive Endoderm and Endoderm Intermediate Human induced pluripotent stem (iPS) cells were differentiated in suspension aggregates using a four (4) stage procedure over approximately two weeks (or 14 days) to generate populations of pancreatic cell types including pancreatic progenitors, endocrine progenitors and hormone-expressing endocrine cells. The human iPS cell line used herein was purchased from S. Yamanaka, Kyoto University, Japan and Cellular Dynamics International, Inc. (CDI).
) provided by

The iPS cells described herein were originally provided by Shinya Yamanaka and subsequently by CDI. Undifferentiated iPS cells were grown in DMEM/F12 containing 20% knockout serum replacement with mitotically inactivated mouse embryonic fibroblasts or preferably feeder-free (without a fibroblast feeder cell layer).
Differentiation was initiated by dissociating undifferentiated iPS cells into single cells using lysine cutase, and cell samples were taken for RNA isolation and analysis. Cells were incubated with insulin-transferrin-selenium (ITS) at a 1:5000 dilution, activin A (100 ng/mL), wn
t3a (50 ng/mL) and the Rho-kinase or ROCK inhibitor, Y-27632
The cells were resuspended at 1-2 million cells per milliliter in RPMI + 0.2% v/v FBS containing 10 μM erythrocyte sedimentation medium and placed in an ultra-low attachment 6-well plate on a rotating platform and spun at approximately 100 rpm. Media was changed daily and cultures were spun at 100 rpm for the remainder of the differentiation process. Growth, passaging and expansion of iPSCs were essentially as described in U.S. Patent Nos. 7,961,402 and 8,211,699.

The methods described herein for generating aggregated suspension cultures of pluripotent cells, e.g., hES or iPS cells, and cells derived from pluripotent cells, are substantially as described in the publication of US Pat. No. 6,333,363 issued Feb. 23, 2007.
The application, entitled Compositions and Methods for Compositions of Compounds, was filed on
ulturing differentiating cells PCT/US2007
/062755, and the application filed on October 20, 2008, entitled Methods a
nd compositions for feeder-free pluripot
ent stem cell media containing human ser
um, as described in PCT/US2008/080516.

The methods described herein can be facilitated by first coating the culture vessel with an extracellular matrix, for example, as described in U.S. Patent No. 6,800,480 to Bodnar et al., assigned to Geron Corporation. As with other methods for culturing other pluripotent stem cells, e.g., hES and iPS cells, this method is substantially similar to that described in U.S. Patent No. 6,800,480, filed Oct. 20, 2008, entitled Methods and Compositions, and entitled "Cell Culture Methods for pluripotent stem cells," which is incorporated herein by reference in its entirety.
ions for feeder-free pluripotent stem
The cells can be cultured using soluble human serum as described in US patent application PCT/US2008/080516, which is entitled "Cell Media Containing Human Serum."

The methods described herein are based on the Wisconsin Alumni Research
As described in U.S. Patent No. 7,005,252 to Thomson, J., assigned to the Ware Foundation (WARF), exogenous fibroblast growth factor (FGF) can be provided from sources other than solely a fibroblast feeder layer.

During the first approximately 24 hours of rotation, the single cells that were attached to each other formed cell aggregates, and the RNA
Sufficient cell samples were obtained for isolation and analysis of iPS cells. The cell aggregates were approximately 60 to 120 microns in diameter. The iPS cell samples were placed on a rotating platform for approximately 1 day (
or 24 hours later, the cultures were treated with 1:5000 dilution of ITS, activin A (100-2
RPMI+ containing 0.000 ng/mL), and Wnt3a (50-100 ng/mL)
0.2% vol/vol FBS for approximately 1 day (day 0 to day 1) and for an additional day;
or in the same medium (days 1 to 2) but without Wnt3a. Daily cell samples were taken for RNA isolation and analysis. After 2 days of differentiation, cultures were treated with 1:100
0 dilution of ITS, KGF (or FGF7, 25 ng/mL) and TGFβ inhibitor no
RPMI + 0.2% volume/volume FBS containing .4 (2.5 μM) for 1 day (or 2
For the next 2 days (days 3 to 5), the iPS cell aggregate suspension was fed with the same growth factor cocktail medium except that the TGFβ inhibitor was omitted from the medium. Again, at the end of this stage (stage 2 or day 5), cell samples were taken for RNA isolation. For stage 3 (days 5 to 8), TTNPB[4-[E
-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid] (3 nM), KAAD-cyclopamine (0.25 μ
The cell culture medium was replaced with DMEM + 1% v/v B27 supplement containing 500 mM NaCl (50 ng/mL) and Noggin (50 ng/mL). Again, at the end of this stage (stage 3 or day 8),
Cell samples were taken for RNA isolation and analysis. For stage 4 (days 8 to 14), noggin (50 ng/mL), KGF (50 ng/mL) and EGF (50 ng/mL) were administered.
The medium was replaced with DMEM containing 1000 mM NaCl + 1% v/v B27 supplement (1 mL/mL). Again, at the end of stage 4 (or day 14), cell samples were taken for RNA isolation and analysis.

Real-time PCR was performed to measure gene expression of various marker genes during differentiation. Gene expression of specific markers or genes was first normalized to the average expression levels of housekeeping genes, cyclophilin G and TATA binding protein (TBP) expression. The normalized relative gene expression was then displayed in a bar graph against the expression levels in undifferentiated iPS cells, thus representing the fold upregulation of various differentiation markers. OCT
For 4, gene expression was normalized to set the lowest sample in the data set (day 14) and therefore represents fold downregulation during differentiation.

2A-L show the expression levels of identified genes (e.g., Oct4, Brachyury, Cer1, GSC, FOXA2, FO) compared to the expression levels of the same genes in undifferentiated iPS cells.
1 is a bar graph showing the relative gene expression of the following genes (HNF6, PDX1, PTF1A, NKX6.1, NGN3, and INS):
The results showed that the gene or expression marker was either up-regulated or down-regulated. For OCT4 (Figure 2A), gene expression was normalized and the sample with the lowest level of expression was set to 1 (1
Thus, as shown by FIG. 2A, the relative expression level of OCT4 was correlated with the fold downregulation (y axis) during differentiation (stages 0 to 4 or days 0 to 14).
axis).

As shown in Figure 2A, OCT4 (POU5F1) is expressed at high levels in undifferentiated iPS cells and shows subsequent downregulation during differentiation (days 0 to 14). By day 14, OCT4 expression levels had decreased more than 3000-fold from those observed in undifferentiated cells. In contrast, the brachyury gene (
There was a transient upregulation of expression of BRACHYURY (Fig. 2B). The transient upregulation of brachyury was the result of directed differentiation of pluripotent/iPS cells to mesoendoderm by application of activin A and wnt3a. As shown by the upregulation of CER1, GSC and FOXA2 by the end of stage 1 on day 3 (Fig. 2C-E).
Endodermal mesoderm was further differentiated into definitive endoderm during days 2 and 3 by continued exposure to Activin A. During stage 2, definitive endoderm was further induced to differentiate into gut endoderm, as indicated by upregulation of FOXA1, maintenance of FOXA2 expression and downregulation of CER1 and GSC by day 6 of differentiation (FIG. 2C-F). During stage 3, exposure to the retinoids, Cyclopamine and Noggin, further induced gut endoderm to differentiate into posterior foregut/PDX1-expressing endoderm, as indicated by upregulation of HNF6 and PDX1 by day 8 (FIG. 2G-H).
As indicated by upregulation of NKX6-1, NGN3, and INS ( Fig. 2I-L ).
During stage 4, exposure to KGF and EGF resulted in the posterior foregut/PDX1-expressing endoderm being further induced to differentiate into pancreatic precursors, endocrine precursors and hormone-expressing endocrine cells.

Example 2
Rho Kinase Inhibitors Promote iPS Cell Growth, Survival, Proliferation and Cell-Cell Adhesion Methods for differentiating various hES and iPS cell lines are substantially as described herein and in Example 1. In addition to the culture conditions described for stages 1, 2, 3, 4 and 5, apoptosis inhibitors and/or Rho kinase or ROCK inhibitors were added to the medium to enhance and promote growth, survival, proliferation and cell-cell adhesion during differentiation. Typically, about 10 μM of a Rho kinase inhibitor, such as Y-27632, was added to the cell culture at each stage. Alternatively, at least stages 1 and 2, and stage 4 were added to the medium.
and 5, or any combination thereof, a Rho kinase inhibitor was added. The morphology and gene marker expression profile of differentiated iPS suspension (aggregate) cell cultures are substantially similar to that of suspension cell cultures derived from hES cells.

3 and 4 show immunocytochemistry (IC) of iPS cell cultures from stages 4 and 5, respectively.
FIG. 3 shows cell aggregates from stage 4 expressing typical gene markers characteristic of PDX1 positive pancreatic endoderm (also called pancreatic epithelium or pancreatic precursors) including PDX1/NKX6.1 co-positive cells. Although not shown in FIG. 3, stage 4 cells do not express hormone secretory proteins or gene markers more characteristic of stage 5 cells such as insulin (INS), glucagon (GCG), somatostatin (SST) and pancreatic polypeptide (PP). FIG. 4 shows cell aggregates of hormone expressing cells from stage 5. These ICC results were further confirmed using QPCR. However,
Because QPCR is a population-wide study of the total levels of RNA expressed in a sample or cell culture,
It cannot definitively show that any one cell expresses multiple markers.

Example 3
Encapsulation of IPS-derived pancreatic precursors To date, methods for generating iPS cells and sources for generating iPS cells have been reported. However, there is no sufficient description of the differentiation of any iPS cells into any functional differentiated cells for potential use in cell therapy to treat certain diseases, such as diabetes.

To determine whether stage 4 PDX1 positive pancreatic endoderm or pancreatic progenitor cell cultures derived from human iPS cells are fully capable of developing and maturing in vivo into glucose sensitive insulin secreting cells, pancreatic progenitor populations substantially as described in Examples 1 and 2 were cultured in the U.S. Pat. App. No. 12/618,618, filed Nov. 13, 2009, and subsequently purified to 100% PBSC.
59. Title ENCAPSULATION OF PANCREATIC LINEAGE
CELLS DELIVERED FROM HUMAN PLURIPOTENT ST
EM CELLS; and U.S. Pat. Nos. 7,534,608 and 7,695,96
No. 5, titled METHODS OF PRODUCING PANCREATIC HORSE
The cells were then added to a macroencapsulation device substantially similar to that described in MONES. Briefly, 5-10-20 μL of gravity-settled cell suspension aggregates having a cell count of approximately 3×10 ⁶ were added to each device.

The encapsulated cells within the device are then prepared for implantation into a mammal, e.g., an immunodeficient SCID/Bg mouse, but may be implanted into larger animals, including rats, pigs, monkeys, or human patients. The method and device for implanting encapsulated cells is substantially as described in U.S. Patent Application Serial No. 12/618,659, U.S. Patent Nos. 7,534,608 and 7,695,965.
No. 6,333,236, which comprises pancreatic progenitor cells implanted in a GELFOAM matrix and transplanted under the epididymal fat pad (EFP).

Although immunosuppression was not required in these studies, it may be required for certain mammals for an initial intermediate period until the precursors in the device are fully matured and responsive to glucose. In some mammals, the immunosuppression regimen may last for about 1, 2, 3, 4, 5 years.
, 6 weeks or more, depending on the mammal.

The transplanted cells were allowed to differentiate and further mature in vivo. For example, human insulin levels are determined by testing the levels of human C-peptide to determine whether the transplanted cells have normal physiological function as naturally occurring beta cells. Human C-peptide is cleaved or processed from human proinsulin, and therefore specific detection of human C-peptide, but not endogenous mouse C-peptide, indicates that insulin secretion is derived from the transplanted (exogenous) cells. The animals with the transplants are tested for human C-peptide levels every 2, 3 or 4 weeks by injecting them with a bolus of arginine or glucose, preferably glucose. The mature beta cells (derived from differentiated pluripotent iPS cells) should then be physiologically functional and responsive to glucose, similar to naturally occurring or endogenous beta cells. In general, amounts of human C-peptide above 50 pM or the average basal (threshold) level are indicative of the function of what are now beta cells from the transplanted precursors.

Kroon et al. (2008), supra, U.S. Patent Application 12/618,659, U.S. Pat.
Encapsulated pancreatic progenitors derived from hIPS cells are expected to mature into functioning pancreatic islet clusters with endocrine, acinar and ductal cells similar to those of naturally occurring islets, as described in U.S. Pat. Nos. 5,534,608; 7,695,965 and 7,993,920. Purified or enriched pancreatic progenitors derived from hIPS cells prior to transplantation are also expected to mature and develop into functioning pancreatic islets and produce insulin in vivo. Specific embodiments for purifying and enriching various differentiated cell populations are described in U.S. patent application Ser. No. 12/107,020, entitled METHODS FOR PARTICULAR PREPARING, filed Apr. 8, 2008.
URIFYING ENDORM AND PANCREATIC ENDORM
M CELLS DERIVED FROM HES CELLS, now U.S. Pat.
Pancreatic progenitors derived from cryopreserved hIPS cells can be thawed and culture-adapted prior to transplantation, thus allowing for in vivo transplantation.
It is further expected that hIP can mature in vivo to produce insulin.
Hypoglycemia can be ameliorated in diabetic-induced animals with transplanted pancreatic progenitors derived from S cells.

In summary, fully encapsulated pancreatic progenitor cells derived from hIPS cells in macroencapsulation devices are expected to mature into physiologically functional pancreatic islets and produce insulin in response to glucose in vivo.

Example 4
Pancreatic Progenitor and Hormone-Secreting Cell Composition Using flow cytometry, differentiated hIPS cell populations were classified into PDX1-positive pancreatic endoderm or pancreatic progenitor cells (stage 4), as shown in Tables 6a, 6b, and 7, respectively.
and their content of endocrine or endocrine precursor/progenitor cells (stage 5). Table 6b is the same data set as Table 6a, but presented similarly to Table 10 for comparison. Overall PDX1+ and NKX6.1+ counts were analyzed for NKX6.1+/PDX1
+/CHGA- cell population (column 5 of Table 6a), the Table 6a populations overlap with each other, e.g., total cell counts exceed 100%. Table 6b includes PDX1+ only and triple negative (residual) data, which are not shown in Table 6a. Certain of these iPEC grafts, as well as others using substantially similar formulations, were implanted into animals to determine in vivo function, although human serum C-peptide levels were not robust enough for any potential therapeutic purposes (data not shown). Values shown are the percentage of total cells belonging to a given population. Pancreatic progenitors (NKX6.1(+)/PDX1(+)) in suspended cell aggregates
The number of NKX6.1+/PDX1-/CHGA- cells and the very small population of NKX6.1+/PDX1-/CHGA- cells were consistent with those observed in pancreatic progenitor cell suspension aggregates derived from hES cells, and are described in U.S. patent application Ser. No. 12/264,760, entitled "Patient Cell Lineage Immunoglobulin Gene Expression and Antigen Expression in Human Embryonic Stem Cells," filed Nov. 4, 2008.
TEM CELL AGGREGATE SUSPENSION COMPOSITION
NS AND METHODS OF DIFFERENTIATION THEREO
The levels of endocrine and/or endocrine precursor/progenitor cells were also analyzed in accordance with U.S. Patent Application Serial No. 12/107,020, filed April 8, 2008, and U.S. Pat.
Title: METHODS FOR PURIFYING ENDODERM AND PAN
CREATIC ENDODERM CELLS DELIVERED FROM HES
The results were substantially consistent with those obtained with hES-derived cell cultures in CELLS. As with hES-derived cell suspension aggregates, varying the concentration of different growth factors in the medium at specific stages of differentiation (e.g., stage 4) should increase and/or decrease specific populations of pancreatic endoderm, endocrine, PDX1-positive endoderm or non-pancreatic cell types.

Example 5
PEC Receptor Tyrosine Kinase The above methods are substantially the same as those described in Table 8 below, which has been adapted from Schulz et al. (2012) supra. These and other methods described herein are not to be construed as limiting the scope of the present invention, but are instead intended to be illustrative, not limiting, and are not intended to limit the scope of the present invention.
Nos. 8,071,825; and 8,153,429.

When applying the above methods to iPS cells and pancreatic progenitors implanted in animals, Applicants observed the same robust iPS cell proliferation and differentiation, as compared to applying the same methods to hESCs and hES-derived pancreatic progenitors.
However, iPS cells have not consistently obtained in vivo function. This was surprising given that iPS cells are human pluripotent stem cells that have hESC morphology and gene expression patterns and can form embryoid bodies in vitro and teratomas in vivo, demonstrating that they can generate cells from all three germ layers. See, at least, e.g., Yu et al. (2007); U.S. Patent Application Publication No. 2009/0047263; International Patent Application Publication No. WO 2005/80598; U.S. Patent Application Publication No. 2008/0233610.
and International Patent Application Publication No. WO 2008/11882, supra. These references describe that iPS cells meet the defining criteria of ESCs. Thus, it has been shown that iPS cells can be used as ESCs in an in vitro differentiation protocol that provides hES-derived pancreatic progenitor cells that further mature and develop in vivo into fully functional glucose-responsive cells.
However, in light of the inconsistent in vivo functional data using the above methods, Applicants believe that the iPSCs may be a surrogate for SCs, as has been consistently observed with pancreatic precursor and/or pancreatic endoderm cells (PECs), i.e., PECs derived from hESCs.
hiPSCs capable of providing substantially similar and robust levels of in vivo function
In this study, we sought to explore a differentiation media formulation that is specific for stage 4 derived cells from iPECs.

Applicants have demonstrated that endocrine (CHGA+ cells) cells present in PEC are multihormonal endocrine cells and that PECs can be used to generate islets with glucose-responsive insulin-secreting cells in vivo.
We have previously reported that the NKX6.1 and PDX-, which are thought to be the PECs that actually generate functioning islets in vivo, are not a subpopulation of cells in C. pleiotropic ovarian hyperplasia (CHD). See Kelly et al. (2011) supra. Rather, it is a non-endocrine cell population (CHGA-cells), specifically the NKX6.1 and PDX-, which are believed to be the PECs that actually generate functioning islets in vivo.
1. Applicants therefore explored whether modulating, altering or shifting the relative ratios of endocrine and non-endocrine subpopulations would affect subsequent in vivo function.

Previous efforts to decipher receptor-ligand signaling in hESCs have been successful in identifying growth factors that promote self-renewal, allowing the development of defined media culture conditions.
(2007). Wang et al. have identified heregulin-β as a ligand that binds to ERBB3 and induces dimerization with ERBB2, thereby affecting hESC self-renewal.
ERBB is a receptor tyrosine kinase (RTK), a widely expressed transmembrane protein that acts as a receptor for growth factors and other extracellular signaling molecules. Upon ligand binding, they undergo tyrosine phosphorylation at specific residues in their cytoplasmic tails, initiating a signaling cascade for the binding of other protein substrates involved in RTK-mediated signaling. RTK functions in several developmental processes, including regulation of cell survival, proliferation and motility, and their role in cancer formation are well documented. ERBB tyrosine kinase receptors were also known to be expressed throughout the developing fetal human pancreas, but the specific roles of particular ERBB receptors and their ligands are unknown. Mari-Anne Huotari et al. (2002) ERBB S
Ignaling Regulates Lineage Determination
of Developing Pancreatic Islet Cells in
Embryonic Organ Culture, Endocrinology 1
43(11):4437-4446.

The self-renewal of pluripotent stem cells in the fetal human pancreas and their expression as demonstrated by Wang et al. (2007) above, and the expression of ERB in ERBB RTK in the human fetal pancreas.
Because of the role of BRTK signaling, Applicants next hypothesize that BRTK signaling may be involved in PEC specification during differentiation, or in expansion by promoting self-renewal, or in vivo expression into physiologically functioning islet hormone-secreting cells.
In an effort to identify receptors and ligands that may enhance some other unknown mechanism that promotes in vivo maturation, we set out to investigate the potential activation of RTKs in in vitro pancreatic endoderm cells (PECs) derived from hESCs. PECs were generated in suspension of differentiating aggregates substantially as described in Table 8, with the following modifications.

Four PEC samples were generated for RTK blot analysis: db-N50 K50
"Steady-state" samples of PEC aggregates at E50 were collected at the end of stage 4 (or d13). "Fasted" samples were cultured in db (DMEM high glucose or 0.5xB-27
The samples represented d12 PEC aggregates that were fed with DMEM high glucose supplemented with supplements (Life Technologies) medium alone (no growth factors) and harvested on d13. Two "pulse" samples were fed and cultured in db medium on d12, then fed with either db-K50 E50 medium or db medium containing 2% FBS for 15 minutes on d13 before harvesting. Such conditions allowed for the detection of RTKs that were active in stage 4 conditions, as well as KG
The aim of the study was to discover what kind of response could be elicited by pulsing with PE, EGF and insulin (present in the B27 supplement) or serum.
We aimed to subject C to broad-spectrum growth factor stimulation, potentially identifying RTKs that are present in C and can be activated but not stimulated in this stage 4 condition.

RTK analysis was performed essentially as previously described in Wang et al. (2007), supra. Briefly, Proteome Profiler™ human phospho-RTK antibody arrays (R&D Systems) were used as per the manufacturer's instructions. 1% NP-40, 2
0 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2
Protein lysates were prepared with 0 mM EDTA, 1.0 mM sodium orthovanadate, 10 μg/mL aprotinin, and 10 μg/mL leupeptin.
Fresh protein lysates of 1000 ng/ml were incubated overnight with nitrocellulose membranes dotted with duplicate spots for 42 anti-RTK antibodies and 5 negative control antibodies, as well as 8 anti-phosphotyrosine positive control spots (Figure 5A). The arrayed antibodies capture the extracellular domains of phosphorylated and non-phosphorylated RTKs, and bound phospho-RTKs are detected with a pan-anti-phosphotyrosine antibody conjugated to horseradish peroxidase (HRP) using chemiluminescence. See Figure 5 for the RTK array layout and Table 9 below for a list of RTKs in the array.

RTK blots (FIG. 6A) showed that the insulin receptor and IGF1 receptor (IR, IGF1R, respectively) were phosphorylated and activated in all conditions, similar to what was previously observed in hESCs. See Wang et al. (2007) supra. The EGF receptor (EGF
ERBB1) was phosphorylated in steady-state conditions, which was expected given the presence of EGF in stage 4 medium. Indeed, low levels of phosphorylation of ERBB2 were detected in both steady-state and fasted conditions. Phosphorylation of both EGFR and ERBB2 was elevated in each pulsed condition, confirming the ability of the assay to detect activation in response to pulsing of ligand. Phosphorylated VEGFR3 was also detected in all conditions and was elevated in pulsed samples. This suggested that PECs generate endogenous VEGFR3 ligands, with possible candidates being VEGF-C and D. Serum pulsing appeared to activate additional receptors, including low levels of ERBB3 phosphorylation. Detection of phosphorylated ERBB2/3 suggests that heregulin-like EGF family members can activate signaling in PEC. TIE-2 is one of two angiopoietin receptors and appeared to be phosphorylated at low levels in response to serum. Angiopoietin-1 and angiopoietin-4 are known to be activating ligands of Tie-2, whereas angiopoietin-2 and angiopoietin-3 function as scene-dependent competitive antagonists. HGF receptor (HGFR) was also phosphorylated in response to serum pulsing, suggesting that hepatocyte growth factor can also elicit signaling in PEC. Finally, low levels of phosphorylation of ephrinB2 RTK (EPHB2) were detected, but ephrin/Eph signaling is a membrane-bound cell-cell signaling system that may not be readily exploited in PEC differentiation. Interestingly, ERBB4 was not phosphorylated. Thus, RTK analysis highlighted several receptors that are phosphorylated in PEC or can be phosphorylated in response to different conditions, e.g., serum. These results suggest that some soluble ligands can elicit RTK signaling in PECs, potentially affecting cell proliferation, differentiation and/or specification and therefore subsequent in vivo maturation into functioning pancreatic islets.

(Example 6)
Heregulin and FGF2 growth factors affect PECs derived from hESC compositions. As described in Example 5 above, certain RTKs are activated (or phosphorylated) under certain conditions.
In view of the RTK analysis demonstrating that ERBB2 and ERBB
Because heregulin 3 appeared to be activated in PEC (after 13 days of differentiation from stages 1-4), applicants sought to determine the effect of heregulin when applied to stage 3 and 4 cells.

Preliminary studies were performed using heregulin and FGF. Certain of these studies included the Rho kinase inhibitor, Y-27632. These preliminary studies were performed using 10 ng/mL
Treatment of pluripotent stem cells at stage 1 for 1 day with mL heregulin-1β (same concentration and heregulin isomer as disclosed in Wang et al. (2007)) reduced heregulin-1β (
The results showed that Hrg1β increased the cell aggregate size of hES-derived cell aggregates in suspension culture compared to the aggregate size of hES-derived cell aggregates in suspension culture without Hrg1β. The increase in cell aggregate size is advantageous in providing a larger cell mass for subsequent transplantation and functional testing in animals. Furthermore, when Hrg1β was increased from 10 ng/mL to 50 ng/mL at stage 3, aggregate disk size increased. This result was also observed when 50 ng/mL of another growth factor, FGF2, was used at stage 3 compared to culture in the absence of FGF2. The increase in cell aggregate size was observed when stage 3 cultures were exposed to FGF2 for additional days, e.g., 2 days.
This was also observed when exposed to 50 ng/mL FGF2 for 3 days compared with 1 day.

Table 10 provides a summary of flow cytometry analysis of PEC cells treated with Hrg1β and FGF2 at stage 3. Endocrine cells are designated as CHGA positive (or CHGA+) cells, and non-endocrine cells are designated as CHGA negative (or CHGA-) cells.
GA+) and non-endocrine cells (CHGA-) may stain positive for other markers, for example staining positive for PDX1 and/or NKX6.1. Cells that do not stain for any of the markers tested are referred to as triple negative or residual cells (CHGA-/NKX6.
1-/PDX1).

To determine whether the increase in cell aggregate size affects the PEC subpopulation,
The composition of the C population was analyzed by flow cytometry.
Compared to control cultures in which 1β and FGF2 were not used, the PEC non-endocrine subpopulation (C
HGA-) increased from 54.01% to 61.2% with the addition of 10 ng/mL Hrg1β at stage 3, and increased from 54.01% to 61.2% with the addition of 50 ng/mL Hrg1β at stage 3.
The endocrine subpopulation (CHGA+) had an HrgO of 10 ng/mL or higher.
Treatment with 1β had little effect, but more so at 50 ng/mL.
The relative levels of residual cells were reduced, more so at 50 ng/mL Hrg1β, and thus the increase in cell aggregate size with Hrg1β treatment was largely attributable to an increase in non-endocrine subpopulations compared with endocrine and residual subpopulations.

The effects of FGF2 in stage 3 cultures were similar, but more pronounced than those of Hrg1β. For example, the PEC non-endocrine subpopulation (CHGA-) was increased, as was Hrg1β. The major effect of FGF2 in these cultures was a substantial reduction in the endocrine subpopulation. In some cases, these cells were nearly undetectable after 3 days of treatment (
32.9% to 0.33%). Thus, the increase in cell aggregate size in FGF2-treated cultures was largely attributable to an increase in non-endocrine, and in some cases residual, cell subpopulations (13.1% to 22.7% over 3 days at stage 3).

Thus, heregulin and/or FGF2 appear to play a role in cell specification in the PEC population, which is surprising given that Wang et al. (2007), supra, reported that heregulin alone plays a role in cell renewal when used in the context of pluripotent stem cells.

(Example 7)
Methods for Improving In Vivo Graft Function of PECs by Treatment of IPS-Derived Cell Cultures with Heregulin Because the methods according to Table 8 when applied to iPSCs to generate iPECs did not provide robust in vivo function in animals, Applicants explored other methods for iPEC generation. Modifications of the standard methods shown in Table 8 include, but are not limited to, the following:
Optimizing SC passage times; modulating levels of BMP signaling; modulating iPSC suspension aggregation parameters during expansion and differentiation (e.g. shear forces,
rotation speed, etc.); optimization of the concentration, timing and duration of use of growth factors, e.g., Wnt, activin and rho kinase inhibitors; and treatment with other growth factors (e.g., ERBB ligands) as candidates to enhance cell mass, proliferation, differentiation, survival, etc., at various stages 1-4 of the differentiation protocol. Many of these iterative experiments were examined alone or in combination to determine how the differentiation method of iPSCs during stages 1-4 could be optimized. Such optimized differentiation methods, when transplanted, were shown to be effective in enhancing the differentiation of hE
The iPEC population generated robust glucose-responsive insulin-secreting cells in vivo similar to those observed and reported for SCs.
We describe baseline conditions with and without heregulin that were demonstrated to differentiate iPSCs into mature iPECs that are glucose-responsive islet cells in vivo.
Similar to those described in Examples 1, 2, and 5 and Table 8 herein, except that heregulin was added at stages 3 and 4. 30 ng/mL of Ηrg1β was used, but concentrations of 10 ng/mL to 50 ng/mL, or even greater than 50 ng/mL, are suitable. Additionally, as described in Example 2, the Rho kinase inhibitor, Y-27632, was used.
The addition of was maintained in differentiation cultures.

To determine the effect of the addition of heregulin or heregulin and a rho kinase inhibitor on stage 3 and 4 cell subpopulations, iPEC populations were analyzed by flow cytometry. Table 12 shows the results of the treatment with the formulations shown in Table 11 and with activin at a concentration of 200 ng/ml.
Table 12 provides a summary of flow cytometry analysis of various iPEC populations using formulations modified by increasing L. Additionally, Table 12 shows the general conditions used in each set of experiments (baseline with and without Heregulin) and the relative percentages of cell types (endocrine, non-endocrine, PDX1 only, and triple negative or residual cell subpopulations) in the iPEC populations. Table 12 also discloses data regarding the in vivo functionality of cells generated in each experiment.

In certain conditions, PECs (hESCs, E2354) and iPECs (E2314, E2
The ratio of cell subpopulations in the 344, E2347) population was altered. For example, sometimes the percentage of endocrine (CHGA+) cells was decreased and the percentage of non-endocrine cells (CHGA-/NKX6.1+/PDX1+) was increased compared to baseline (no heregulin) conditions. Experiment #2
380 (E2380), heregulin appeared to be responsible for the change in the proportion of endocrine cells compared with non-endocrine cells in these PEC and iPEC populations, although the level of endocrine (CHGA+) cells was increased, not decreased, by addition of heregulin.

To determine whether changes in the composition of the PEC and iPEC populations affected in vivo function, PEC and iPEC grafts from most of the experiments described in Table 12 were implanted into mice substantially as previously described herein and in Schulz et al. (2012) and Kroon et al. (2008) supra, and in Applicant's other patent and non-patent publications, including U.S. Pat. Nos. 7,534,608; 7,695,965; 7,993,920 and 8,278,106 supra. Briefly, the PEC and iPEC populations were all encapsulated in biodegradable, semi-permeable cell encapsulation devices, some of which contained microperforations. The devices were manufactured by Applicant and described in U.S. Pat. No. 8,278,106, filed Nov. 13, 2009, entitled ENCAPSULATION OF PANCREATIC CARCINOMA AND ITS USE IN HUMAN ...
CELLS FROM HUMAN PLURIPOTENT STEM CELLS
The glucose stimulated insulin secretion (GSIS) assay was performed from about 56 days after transplantation. Blood was collected before (fasting) and combinations of 30 and/or 60 minutes after glucose challenge. Graft function was assessed by measuring serum human C-peptide concentrations in response to glucose challenge.

The amount of human C-peptide released into the serum is an indicator of the amount of insulin released.
The peptide is a short 31 amino acid peptide that connects or links the A and B chains of proinsulin and preproinsulin, and is secreted by functioning beta or insulin secreting cells. As previously described by Kroon et al. (2008) and others, measurement of human C-peptide is suitable for assessing the release of newly produced insulin by transplanted cells. Thus, the levels of human C-peptide in the serum of these animals are consistent with the release of mature PEC and iPEC.
C-peptide is a measure of in vivo function of the graft. Human C-peptide was detected in serum by at least 8 weeks after transplantation. Additional weeks of transplantation and fasting increased glucose-stimulated C-peptide levels, shifting the peak C-peptide level from 60 minutes to 30 minutes after glucose challenge, indicative of a faster response to glucose challenge following insulin cell maturation. There were a few mice that failed to demonstrate function or were sacrificed due to poor health. However, these mice were in a cohort that otherwise demonstrated high-functioning animals, thus suggesting graft failure rather than an inability of the transplanted cells to differentiate and function.

7A-C show human C-peptide levels in serum following glucose challenge for all of the experiments shown in Table 12 except E2344. FIGS. 7A-C show that grafts resulting from heregulin treatment generally had higher levels of human C-peptide in serum compared to baseline controls. For example, in FIG. 7A, in experiment 2380, there is an approximately 5-fold increase (933 pM vs. 200 pM 60 minutes after glucose challenge) in grafts resulting from heregulin treatment compared to those prepared without heregulin (baseline).
FIG. 7C) Serum C in grafts resulting from heregulin treatment compared to baseline controls
Heregulin does not show higher levels of peptides, suggesting that PE produced from hESCs
Furthermore, PECs derived from hESCs (Cy
T203) and iPEC derived from iPSCs, the iPEC grafts were
The iPSCs have the same functions as the transplants in vivo (see, for example, FIG. 7A and FIG. 7B (iPSCs
Compare Fig. 7C (CyT203 hESCs) with Fig. 7C (CyT203 hESCs). Thus, iPEC grafts are as robust as PEC grafts. Furthermore, iPECs from E2380, which did not have the same shift in endocrine and non-endocrine subpopulations, also showed superior function, suggesting that iPECs
The relative ratio of endocrine to non-endocrine cells, which appeared to affect some of the populations (eg, E2314, E2347 and E2354), did not appear to affect in vivo function (see Figures 7A-C).

In addition to testing glucose-stimulated insulin secretion, mature iPEC grafts were tested to determine whether they alone could maintain euglycemia similar to that maintained by hESC-derived PECs when the host animal's beta cells were destroyed. This required destroying the beta cells of the transplanted mice using streptozotocin (STZ), a beta cell toxin that exhibits greater cytotoxicity to mouse beta cells compared to human beta cells. Random non-fasting blood glucose was measured for each mouse before and after STZ treatment. Explantation of the iPEC grafts 13 days after STZ treatment resulted in the resumption of hyperglycemia (noted by a spike in blood glucose), demonstrating control of glycemia by the iPEC grafts and not the endogenous mouse pancreas (see Figures 8A and 8B).

Furthermore, there appeared to be a synergistic effect when heregulin and a rho kinase inhibitor were provided during stages 1-4 of differentiation (see Table 11). For example, iPSCs treated with heregulin at stages 3 and 4 without a rho kinase inhibitor resulted in visibly inferior cell mass, making transplantation impossible. Further supporting evidence of heregulin and rho kinase inhibitor synergy was evident in some of the experiments, e.g., E2356, E2380, where baseline conditions with rho kinase inhibitor alone did not perform as robustly as transplants with rho kinase inhibitor and heregulin (Figures 7A and B).
See Table 1). Treatment with heregulin and the rho kinase inhibitor did not appear to be additive, as the addition of heregulin alone provided cell masses that were insufficient for engraftment and the addition of the rho kinase inhibitor alone (baseline conditions) had poor in vivo function. Thus, providing heregulin alone or the rho kinase inhibitor alone did not substantially resemble the sum of the effects of the two combined; i.e., neither alone provided robust glucose responsiveness in vivo, but in combination they provided glucose responsiveness similar to that of hES-derived cells. Thus, providing both heregulin and the rho kinase inhibitor appeared to be synergistic, as their combined effect was greater than the sum of the effects of each separately. i.e., iPECs treated with rho kinase inhibitor and heregulin matured in vivo, exhibited glucose-stimulated insulin secretion, and were able to maintain normoglycemia in a diabetic mouse model (see Figures 7A-B and 8A-B).

ERBB functionality requires ligand binding, receptor dimerization and receptor trafficking. Variability in each process can result in differential regulation of the receptors and the downstream signals they control. For example, different ERBB ligands bind to ERBB receptors with different affinities, thereby altering the pattern and dynamics of ERBB dimer organization. Table 13
shows many possible different combinations of ligand and receptor binding complexes. A review of the complexity of this system is given in Oda et al. (2005) A comprehensive pattern
Hway map of epidermal growth factor recipe
ptor signaling, Mol. Syst. Biol., 1 (2005) and Lazzara et al. (2009) Quantitative modeling pers.
objectives on the ERBB system of cell regulation
Laboratory processes, Experimental Cell Research
rch 315(4):717-725.

Huotari et al. suggested that neuregulin-4 can modulate the relative levels of endocrine cell subpopulations by increasing the number of somatostatin (delta) cells at the expense of glucagon (alpha) cells, and that neuregulin-4 does not affect the ratio of exocrine (e.g., amylase) to endocrine (e.g., β-insulin, α-glucagon, δ-somatostatin, PP-pancreatic polypeptide) cells. However, these studies were performed by incubating neuregulin-4 on whole mount organotypic tissue cultures obtained from mice at day E12.5. These mouse explant cell populations were cultured at stage 3 (e.g., PDX1-negative foregut endoderm) as described herein.
and/or stage 4 (PDX1-positive foregut endoderm) cell populations. Since neuregulin-4 binds only to the ERBB4 RTK, only endocrine subpopulations of whole mount mouse cultures can be modulated by neuregulin-4 in this context. Thus, since Hrg1 has previously been shown to bind to ERBB3 and induce ERBB2/3 dimerization, the differentiation of stage 3 (PDX1-negative foregut endoderm) and/or stage 4 (PDX1-positive foregut endoderm) cell populations as described herein with a different ERBB ligand, e.g., Hrg1, may be possible.
Treatment of (PDX1-positive foregut endoderm) cells is not expected to modulate the relative endocrine subpopulations as in Huotari. However, as shown in FIG. 6, E in PECs
Due to the low level expression of ERBB2 and 3, it was unclear whether stage 3 and 4 cells express low or high levels of ERBB2 and 3 and bind Hrg1.

Furthermore, on a different occasion, Applicants have described that Hrg1 binds to ERBB2/3 and promotes self-renewal of pluripotent stem cells (see Wang et al. (2007)).
While it is possible that heregulin could act in the same capacity in the context of stages 3 and 4, applicants have previously noted that the majority of cell expansion for the generation of PECs occurs at the pluripotent stem cell stage (stage 0). During stage 0, hESCs are grown, passaged, and expanded for approximately two (2) weeks. Thus, most of the cell expansion or self-renewal that results in cell expansion does not occur during stages 1-4. See Schulz et al. (2012), supra. Furthermore, given that ERBB2/3 is present during stages 3 and 4, one might expect heregulin to have a similar effect on pluripotent stem cells (self-renewal) as opposed to influencing induced differentiation. The functional differences then appear to be dependent on the context, i.e., pluripotent stem cells versus cell types of endoderm or pancreatic lineage.

In summary, in vitro provision of heregulin or heregulin and a rho-kinase inhibitor to foregut endoderm (stage 3) and PDX1-expressing pancreatic endoderm cells (end of stages 3 and 4) generated PEC and iPEC populations that matured in vivo when transplanted and developed into glucose-responsive insulin-secreting cells (see Figures 7 and 8). Such use of heregulin or heregulin and a rho-kinase inhibitor is reported here for the first time. Such uses and effects are indistinguishable from those previously described in patent or non-patent literature.

(Example 8)
Activin Suppresses NGN3 Expression and Generation of Endocrine Lineage-Committed Cells in Pancreatic Endodermal Cell (PEC) Cultures Pancreatic endoderm cells, or "PECs," are a cell population of the pancreas that is primarily composed of two distinct subpopulations: (i) a non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-), which develops and matures in vivo to generate glucose-responsive insulin-secreting beta cells (hereafter referred to as "PECs").
(ii) a subpopulation of cells committed to the endocrine lineage (CHGA+), which can generate other non-insulin-secreting cells or islet supporting cells during in vivo maturation (hereafter "endocrine (CHGA+)"). The non-endocrine (CHGA-) subpopulation is CHGA negative and predominantly positive for PDX1 and NKX6.1, whereas the subpopulation of cells committed to the endocrine lineage is CHGA positive and largely negative for PDX1 and NKX6.1. The non-endocrine (CHGA-) subpopulation is characterized by a lack of systemic detection of insulin responses in vivo (
It takes approximately 8-12 weeks or more for the primary PECs to develop and mature into glucose-responsive insulin-secreting beta cells in amounts that allow for endocrine proliferation (as assayed by ELISA for C-peptide). However, it takes less than 8 weeks for glucose-responsive cells to develop and mature in vivo during normal embryonic development. Thus, it remains desirable to obtain: (1) a PEC population with an increased non-endocrine (CHGA-) subpopulation; and/or (2) an in vivo
A true endocrine (CHGA+) cell population that is glucose responsive in vitro and/or a developmentally advanced population (e.g., well-specified and has specific signature markers consistent with those described for similar pancreatic populations in vivo), and/or (3
2.) A progenitor cell population capable of shortening and/or reducing the time it takes for development and maturation in vivo.

With respect to the first goal, it is desirable to obtain a PEC population in which differentiation into endocrine lineage-committed cells (CHGA+) is suppressed similar to that which occurs naturally in vivo, while the non-endocrine (CHGA-) subpopulation remains intact.
The method for generating the population is described below.

During normal in vivo development, the transcription factor neurogenin-3 (NGN3) is a master regulator of endocrine cell development, and expression of NGN3 is thought not to occur until after upregulation of PDX1 and NKX6.1 in, for example, pancreatic endoderm lineage cells in vivo. Rukstalis and Habener (2009), Neurogenin
3: A master regulator of panic islet
differentiation and regeneration, Islets
1(3):177-184. In accordance with Table 8, retinoic acid (RA) or a retinoid analog, 4-[(E)-2-
(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)
In this study, retinoic acid is used to induce endocrine cell lineage (CHGA+) expression in PECs. However, retinoic acid also induces NGN3 expression early, which initiates endocrine specification. Thus, suppression, repression or inhibition of NGN3 during stage 3 and/or stage 4 may provide a clue to minimize cells committed to the endocrine lineage (CHGA+) subpopulation in PECs and/or to obtain endocrine cells later in development in vitro and/or in vivo. Importantly, NGN3
Any suppressor, repressor or inhibitor of
) should not be suppressed, repressed or inhibited, and preferably this subpopulation should be expanded during the in vitro generation of PECs.

Various growth factors were investigated for their ability to repress genes such as NGN3 during stages 3 and 4. One candidate growth factor was activin, which has multiple functions in stem cell expansion and differentiation. For example, at low levels (e.g., 10 ng/mL), activin helps maintain pluripotency and cell proliferation of pluripotent stem cells. At higher levels (
At, e.g., 100 ng/mL), activin acts as a differentiation growth factor responsible for stage 1 definitive endoderm generation (see, e.g., Table 8).
To test its effect on suppression of cells committed to stage 3, applicants first tested Activin at a median concentration of approximately 50 ng/mL in standard adherent differentiation. At this level, Activin (ACT) was able to suppress NGN3 expression without suppressing PDX1 and NKX6.1 gene expression in PECs (compare Figures 9D-E with Figures 9A-B). However, when the same level of Activin was used in the more scalable cell aggregate suspension cultures during stage 3, the total cell mass was greatly reduced and in some cases the cell aggregates disappeared (compare control and Activin-treated cell aggregates in Figure 10).
A similar reduction in cell aggregates was observed with 25 ng/mL activin as well (data not shown).

In light of these results, lower concentrations of activin (e.g., 5 ng/mL and 10
ng/mL) were tested at stages 3 and 4. As shown in FIG. 11, at lower activin concentrations, cell aggregates were able to maintain cell mass and yield compared to controls (FIG. 11B-C at day 8 and FIG. 11B-D at day 12, and FIG. 11C at day 8 and day 12, respectively).
(Compare to control in A). Thus, activin levels below about 25 ng/mL are acceptable to maintain cell mass and yield. To determine whether low levels of activin during stages 3 and 4 affected cell aggregate composition, Applicants next performed flow cytometry analysis on these cell cultures. See Table 14. Table 14 shows:
We show that low levels of activin do not reduce the generation of the non-endocrine (CHGA-) subpopulation, and that when treatment is applied at both stages 3 and 4, it can actually increase this subpopulation (see results for Stg (Stage) 3 & 4 in Table 14 A5). However, the very similar percentages of endocrine (CGA-) cells seen with and without activin are not significantly different from those seen with activin.
Considering the NKX2.2+ (HGA+) cells, differentiation into cells committed to the endocrine lineage (CHGA+) appears to still be significantly induced (see Endocrine column, Table 13). Thus, although Activin can suppress or inhibit NGN3 and NKX2.2 expression as observed by QPCR, at lower levels (allowing for maintenance of cell mass), it does not reduce the endocrine lineage (CHGA+) committed subpopulation when analyzed at the end of stage 4. Advantageously, particularly when Activin is used at stages 3 and 4, there is a significant reduction in PDX1 expression compared to controls.
The single and triple negative subpopulations are reduced.

(Example 9)
Activin, Heregulin and WNT suppress the generation of cells committed to the endocrine lineage in PEC cultures. Example 8 shows that moderate levels of Activin (e.g., 50 ng/ml) during stages 3 and 4 suppress the generation of cells committed to the endocrine lineage.
These results show that Activin (L) was able to suppress or repress NGN3 expression (FIG. 9), but these same levels did not maintain cell mass. Lower levels of Activin (e.g., 5 and 10 ng/mL) were able to maintain cell mass (FIG. 11), but were not sufficient to continue to repress endocrine differentiation during stage 4, as the percentage of endocrine lineage cells (CHGA+) remained substantially similar to that described in the control (Table 14, CHGA+ column) and previous examples. Thus, these low levels do not appear to be sufficient to repress differentiation into cells committed to the endocrine lineage (CHGA+), primarily during stage 3, as continued repression of differentiation of this subpopulation through stage 4 appears to still be necessary. See Rukstalis et al. (2009), supra. Therefore, we sought additional growth factors that could prevent cell loss and yield (i.e., counteract the effects of moderate to high Activin levels) while simultaneously not affecting the generation of non-endocrine multipotent pancreatic progenitor subpopulations and suppressing differentiation of endocrine lineage-committed cells (CHGA+).

Heregulin increased PEC endocrinology (CHGA+) at 30 ng/mL during stages 3 and 4.
Given the above examples describing how reducing the concentration of heregulin when combined with activin often alters the relative levels of endocrine (CHGA-) and non-endocrine (CHGA-) subpopulations, Applicants examined what effect reducing the concentration of heregulin might have on PEC generation when combined with activin.

Pluripotent stem cells were differentiated using a standard protocol according to Table 8, but during stage 3,
Activin was included at 10 ng/mL or 20 ng/mL along with heregulin at 2 ng/mL or 10 ng/mL; during stage 4, activin was reduced to 5 ng/mL and heregulin at 1 ng/mL for 2 days and then at 10 ng/mL.
As shown in the images from day 1 (upper panel), 10 ng/mL activin and 2 ng/mL
Heregulin at 2 ng/mL showed some reduction in cells compared to the control (compare Figures 12A and B, top panel).
Increasing the concentration of α-glucose exacerbated cell loss (compare Fig. 12B and C, upper panels).
However, increasing heregulin to 10 ng/mL in the context of 20 ng/mL activin was able to prevent the greater cell loss at higher concentrations of activin (20 ng/mL) (compare Figures 12D and C, top panel). Thus, while significant cell loss is observed when heregulin levels are too low, upon increasing heregulin levels, for example at least at 20 ng/mL, there is a shift in the percentage of non-endocrine cells (CHGA-) compared to the endocrine lineage (CHGA+) subpopulation.

Quantitative PCR analysis was performed to determine the relative levels of gene expression at days 8, 10, and 12 of differentiation with the above combinations of activin and heregulin (Figure 13). Similar to what was shown in Example 8 (Figure 9), activin at 10 and 20 ng/mL increased NKX
It was possible to suppress NGN3 and NKX2.2 (another endocrine differentiation marker) without reducing NKX6.1 expression (compare Figure 13A and B with C).
The fact that expression remains high indicates that the generation of the non-endocrine (CHGA-) subpopulation remains unchanged or unaffected.
10 ng/mL heregulin, 25 ng/mL activin, and 50 ng/mL activin (Figure 10
) the activin-dependent cell loss previously observed when IgG4 was used in combination with IgG4 was greatly reduced (
(Figure 12, D and E, upper panels). Thus, heregulin appears to effectively counteract the cell loss initially observed with the use of activin at stage 3.
Although N3 and NKX2.2 induction still occurs (i.e., it is not as strongly suppressed as observed when 50 ng/mL activin was used at stages 3 and 4 in adhesion-based differentiation in Example 8, FIG. 9), it is significantly reduced compared to standard conditions (FIG. 1).
3A and B, compare the control condition (Con) and the A20H10 condition).

Activin above 20 ng/mL would further repress the markers NGN3 and NKX2.2 and therefore further endocrine differentiation, but higher levels of heregulin would then be required to maintain cell mass to offset the effects of administering high concentrations of activin. Even higher levels of heregulin as observed in the examples above
The relative production of endocrine (CHGA+) and non-endocrine (CHGA-) subpopulations of PECs can be altered. Furthermore, the effect of higher levels of activin on the repression of NGN3 expression was still required. Thus, Applicants have demonstrated that the expression of activin in the blastocyst-derived blastocysts can be enhanced by, but is not limited to, the induction of anterior and posterior endoderm proliferation and can control other aspects of development.
NT, FGF, PDGF, retinoic acid (RA or TTNPB), BMP, hedge
Additional growth factors or morphogens were explored, including hog, EGF, IGF, etc.

Specifically, Wn was added to activin and heregulin differentiation medium at stages 3 and/or 4.
Addition of t3A was investigated to affect the canonical Wnt signaling pathway. Again, no combination affected the non-endocrine multipotent pancreatic progenitor subpopulation while suppressing differentiation of cells committed to the endocrine lineage (CHGA+) as observed through the repression of NGN3 and NKX2.2 expression, and maintaining superior cell mass (i.e.
One such mixture tested was 50 ng/mL activin along with 10 ng/mL heregulin and 2 ng/mL heregulin at stage 3.
of Wnt; and as above, 5 ng/mL activin and 1 ng/mL activin during stage 4.
/mL heregulin for two (2) days, followed by 5 ng/mL activin and 10 ng/mL
/mL heregulin. This resulted in a larger cell (PEC) mass than the same concentrations of activin and heregulin alone (compare Figures 12D and E, top panels), and an even larger cell mass than the control (compare Figures 12A and E, top panels). QPCR analysis of cell aggregates under these conditions (e.g., combination of activin, heregulin and Wnt at stage 3, and activin and heregulin at stage 4) showed that
NGN3 and NKX2 at stages 3 and 4, especially days 8, 10 and 12.
As observed by the decreased expression of CHGA+.
) showed that there was a significant suppression of differentiation-committed cells. Furthermore, NKX6.1 expression was
Although initially suppressed at day 8, it was significantly elevated by days 10 and 12 (stage 4) (FIG. 13C). Thus, the development of non-endocrine (CHGA-) subpopulations was not impaired by addition of Wnt, whereas differentiation of cells committed to the endocrine lineage (CHGA+) appeared to be delayed or inhibited, without any apparent loss of cell mass.

To determine the cellular composition of the PEC population with the combination of activin, heregulin and Wnt during stage 3, flow cytometry was performed after stage 4 and the PEC cellular composition is shown in Table 14. As shown, the combined growth factors (activin, heregulin and Wnt)
nt, or "AHW"), as observed by a reduced percentage of that subpopulation (15.6 vs. 58.8) compared to control cell cultures under standard protocols,
The results of the study showed that the 3-HTLV-1 agonist, β-lactamase, effectively reduced differentiation of cells committed to endocrine endocrine (CHGA+) but increased the non-endocrine (CHGA-) subpopulation (58.1 vs. 23.4) (Table 8).
This supports and is consistent with the data from QPCR, where the combination of two factors (AHW) generally delayed or inhibited differentiation of cells committed to the endocrine lineage (CHGA+) of PECs without affecting the generation of the non-endocrine (CHGA-) subpopulation. The delayed expression of these genes in vitro is consistent with their onset (or delay) in mammalian pancreatic development in vivo, where NGN3 expression in endocrine differentiation occurs after the onset of PDX1 and NKX6.1 expression in the non-endocrine (CHGA-) subpopulation. Furthermore, there was no impairment in PEC mass and yield.

Given the results using the combination of activin, heregulin and Wnt, activin levels were titrated again in stage 3 to establish the concentration range in which activin could be used. Activin concentrations tested were approximately 0, 25, 50, 75 and 100 ng/mL during stage 3, Wnt at 50 ng/mL and heregulin at 5 ng/mL.
During stage 4, activin decreased to 5 ng/mL, heregulin decreased to 5 ng/mL, and W
nt were not included. Cells were imaged at the end of stage 4 (day 13) and are shown in Figure 12 (lower panel). At higher activin concentrations, e.g., 75-100 ng/mL, cell loss was again evident (Figure 12D and E, lower panels). Importantly, WN at stage 3
The incorporation of T allows for the use of much higher concentrations of activin at stage 3 than would otherwise be possible due to cytoreduction.

To test the effect of sequential activin and heregulin during stage 4 in the context of activin, heregulin and Wnt at stage 3, human ESCs were differentiated using three conditions: (i) standard protocol (see Table 8; or protocol number 1 in Table 17); (ii) activin (75 ng/mL), heregulin (5 ng/mL) at stage 3;
/mL) and Wnt (50 ng/mL) followed by standard stage 4 factors; and (iii) Activin (75 ng/mL), Heregulin (5 ng/mL) at stage 3;
A combination of Activin (5 ng/mL) and Wnt (50 ng/mL) was administered, followed by additional Activin (5 ng/mL) and Heregulin (5 ng/mL) during stage 4, see Table 17. Flow cytometry was performed after stage 4 for all three conditions, and the PEC cell composition was compared as shown in Table 1.
7. AHW at stage 3 reduced cells committed to the endocrine lineage and increased the non-endocrine (CHGA-) subpopulation content compared to controls (21.8 vs. 42.2 for cells committed to the endocrine lineage subpopulation; 40.1 vs. 2 for the non-endocrine (CHGA-) subpopulation).
7.6), and additional treatment with AH at stage 4 further reduced endocrine lineage-committed cells and further increased the non-endocrine (CHGA-) subpopulation content (6.46 vs. 21.8 for endocrine lineage-committed cells; 7.48 vs. 21.8 for non-endocrine (CHGA-) paternal subpopulation).
2.7 vs. 40.1).

Example 10
PEC grafts generated with a combination of activin, heregulin and WNT were
Example 9 shows that the combination of activin, heregulin and Wnt ("APC") improved glucose response function in vivo based on morphology (e.g., low magnification microscopy of differentiated cell aggregates), QPCR and flow cytometry data.
HW") without simultaneously having any adverse effects or improvements on cell mass or yield, and
We demonstrated that AHW-derived PEC aggregates appear to be able to effectively suppress cells committed to endocrine lineage (CHGA+) differentiation, as observed by suppression of NKX2.2 and NKX2.3 expression, and maintain or increase the non-endocrine (CHGA-) subpopulation. To determine whether these changes affect the ultimate development and maturation of glucose-responsive insulin-secreting cells, AHW-derived PEC aggregates were transplanted into animals along with PECs generated from a standard protocol according to Table 8 or from protocol no. 1 in Table 17.

Specifically, human pluripotent stem cells were differentiated using a combination of three factors (AHW) at stage 3, followed by activin and heregulin alone at stage 4. Control PECs were generated substantially according to Table 8. Cell aggregates were analyzed by QPCR at days 8, 10, 12 and 14, as shown in Figure 14. Both the control (Con) and modified AHW protocols generated PECs as observed by NKX6.1 expression (Figure 14A), but only activin and heregulin were differentiated at day 14.
There is more robust NKX6.1 expression using AHW on day 8 (after stage 4). At the same time, differentiation of cells committed to the endocrine lineage (CHGA+) was highly suppressed with the AHW protocol compared to the control protocol, as observed by the suppression of NGN3 expression, especially on day 8 (Figure 14B).

On day 14 of differentiation, PEC aggregates from both methods were packed into Applicant's proprietary implantable semi-permeable encapsulation devices and transplanted into SCID beige mice essentially as previously described (
(n=5 animals for each of the AHW and control methods, total n=10.) Eleven weeks after transplantation, PECs generated using the AHW protocol were induced 1 hour (60 min) after glucose administration.
After the treatment, C-peptide levels averaged approximately 900 pM, whereas control PECs had C-peptide levels of 400 pM.
See Figure 15, which compares AHW animals with controls.
After one week, three of five mice with AHW grafts had an average of 10% human C-peptide.
200 pM or higher (Figure 15, animal numbers 3514, 3516 and 3518), whereas the levels of C-peptide in control animals were significantly lower. In fact, only one of five control animals had levels approaching 600 pM (Figure 15, animal number 3524). Thus,
Based on C-peptide levels, 11 weeks after transplantation, the AHW protocol was able to generate a PEC population with at least two-fold improved in vivo function as measured by levels of human C-peptide after glucose stimulation, potentially correlating with a faster maturation time.

Fourteen weeks after transplantation, AHW PEC grafts continued to outperform control grafts,
The insulin secretion response time was faster, as C-peptide values 30 minutes after glucose challenge for the HW PEC grafts were higher than those 60 minutes after challenge for the control grafts (Figure 16). Specifically, 3 of 4 animals with AHW PEC grafts had C-peptide averaging 3500 pM or higher 30 minutes after glucose challenge, compared to the top 3 control animals, which averaged approximately 2300 pM 60 minutes after glucose challenge (Figure 17).
(Figure 16) This data indicates that differentiation using a combination of three growth factors (AHW) outperformed at the time points tested (i.e., increased serum C-peptide levels 30 minutes after glucose challenge, indicating faster glucose responsiveness from transplanted AHW-derived PEC grafts) or
This demonstrates that it is possible to generate PEC populations that are potentially more potent than standard PEC protocols. Without limiting this application to any one theory, it is believed that the increased potency of AHW PEC grafts is attributable to the fact that the AHW combination (1) delayed or suppressed differentiation of cells committed to the endocrine lineage (CHGA+) in the PEC, (2) increased the non-endocrine multipotent pancreatic progenitor subpopulation in the PEC, and (3) maintained superior cell mass.

(Example 11)
In Vitro Culture of Pancreatic Endocrine Cells Examples 9 and 10 describe the deliberate delay, suppression, inhibition or inhibition of differentiation of cells committed to the endocrine lineage (CHGA+) at stages 3 and 4. In an effort to generate endocrine cell precursors or endocrine cells at advanced stages in vitro, Applicants have cultured cells at stage 5.
Various methods were tested during the study, including 6 and/or 7. For example, Applicants tested which growth factors, alone or in combination, were capable of stimulating or inducing endocrine differentiation, i.e., inducing NGN3 expression.

Table 17 below shows the standard protocol (protocol number 1) for generating PEC and the AHW
Various methods for endocrine generation are summarized along with the protocol (Protocol No. 2) used to generate PECs. However, Applicant has tested, and one of skill in the art will understand, that Table 17, Protocol No. 3, lists several growth factors at specific days and stages, and that not all growth factors, alone or in combination, need to be used on the exact days/stages indicated. Thus, Table 17 represents a number of iterations (e.g., growth factors, duration, etc.) that have been tested by Applicant.

Previously, U.S. Patent No. 8,129,182, issued March 6, 2012, ENDOC
RINE PROGENITOR/PRECURSOR CELLS, PANCREAT
IC HORMONE EXPRESING CELLS AND METHODS O
In F PRODUCTION, Applicants disclosed the use of gamma secretase inhibitors for the generation of endocrine precursors/progenitors. As described and claimed in the '182 patent, Notch 1 is used to further induce PDX1-positive pancreatic endoderm cell differentiation into the endocrine lineage.
Growth factors or agents that inhibit signal transduction have been identified, including inhibitors of gamma secretase (e.g., DAPT or N-[N-(3,5-difluorophenylacetyl)
This was achieved by application of the gamma secretase inhibitor [Gamma Secretase Inhibitor] (Gamma Secretase Inhibitor) (Gamma Secretase Inhibitor) (Gamma Secretase Inhibitor) (L-alanyl-S-phenylglycine t-butyl ester and related analogues). Gamma secretase inhibitors block intramembrane cleavage of the Notch molecule, thereby preventing release of the activated Notch intracellular domain. To induce NGN3 expression, for example, a 2-4 day application of the gamma secretase inhibitor DAPT on the last day of retinoic acid addition or immediately after withdrawal of retinoic acid was used. Here, another gamma secretase inhibitor, RO44929097, was used at stage 5. See Table 17.

As shown in Table 17, during stages 6 and 7, treatment with agents such as, but not limited to, BMP4 ("BMP"), Sonic
Hedgehog ("SHH"), platelet-derived growth factor ("PDGF"), FGF2 (
"FGF"), retinoic acid and/or retinoic acid analogs, such as TTNPB ("
"TTNPB"), insulin-like growth factor I (IGF-I or "IGF1") and -I
I (IGF-II or "IGF2"), vitamin B3 derivatives, nicotinamide ("NC
") or a combination of one or more of these growth factors was applied. One such combination is described in Table 17, Protocol No. 3.

For example, PECs generated from the AHW protocol substantially as described above in Example 10 may be cultured according to Table 17, Protocol No. 3, with 1 μM of a gamma secretase inhibitor (RO4929097 ("
This stage 5 treatment results in the induction of NGN3 expression and subsequent endocrine differentiation.
Nicotinamide was included during stages 5 and 6. NGN3 gene expression was analyzed by Nanostring detection of mRNA after stage 4 (day 13 of culture), after stage 5 (day 15 of culture), and 2 days after stage 6. As shown in Figure 17, gamma secretase inhibitors strongly induced NGN3 expression after stage 5 on day 15, compared to the low level expression of NGN3 at the end of stage 4 (day 13). Following removal of gamma secretase inhibitors on day 15 (the first day of stage 6), NGN3 expression increased as observed on day 17.
GN3 expression is decreased (FIG. 17). This transient increase in NGN3 expression during stage 5 is consistent with its transient expression observed during beta cell development in vivo.
See Jorgensen et al. (2007) supra.

The total number of days for any of the stages can and does vary, for example stages 5, 6 and/or 7 can be shorter or longer in number of days, and this applies to the earlier stages 1, 2, 3 and 4 as well.

Additionally, to promote cell survival and cell mass and yield, stages 1, 2, 3,
A rho kinase inhibitor may be added to the differentiation medium continuously or intermittently during any of stages 4, 5, 6 and 7. Rho kinase inhibitors appear to promote differentiation of induced pluripotent stem cells, e.g., at stages 1, 2 and 4, e.g., by maintaining cell mass and yield.

Furthermore, at about day 20 (i.e., the junction between stages 6 and 7 of protocol number 3 in Table 17), the cell aggregates can be dissociated and reaggregated. This dissociation and reaggregation removes specific cell subpopulations, thus primarily endocrine and/or endocrine precursor/progenitor/end ...
The progenitor (CHGA+) cell population was left behind. This physical manipulation also facilitated the analysis of endocrine populations,
Purification steps are configured as required, see Example 14 for details.

Additionally, starting from stage 6 to stage 7, Matrigel may be used at very low concentrations, e.g., about 0.05%, as needed, to help maintain the cell aggregates and/or prevent endocrine cells from migrating out of the cell aggregates. The use of Matrigel or any other composite cell matrix may depend on the particular cell aggregates themselves (e.g., PEC aggregates, endocrine precursor/precursor cell aggregates, etc., as described below), and potentially the PSC lineage from which the cell aggregates were derived. Other composite or defined matrices, such as serum, laminin, fibronectin, collagen, and other extracellular matrix proteins, may potentially be used to perform substantially similar functions. See Example 14 for details.

At stage 6 or 7, or during stage 6, e.g., day 16, 17 or 18
Beginning on day 17, insulin-like growth factor (IGF) 1 and/or 2 (IGF1 or IGF2) may be added. Pluripotent stem cells were differentiated substantially as described above according to Table 17, except that during stage 6, specifically on day 17, insulin-like growth factor (IGF) 1 and/or 2 (IGF1 or IGF2) was added at 50 or 200 ng/m
L of IGF1, or 25 or 100 ng/mL IGF2 were added to the cell cultures. After stage 6 (day 20), cell aggregates were analyzed for mRNA using Nanostring. These preliminary studies showed that IGF2 was more capable of inducing INS expression than IGF1 (data not shown), but longer treatments could produce more robust effects from IGF2 alone or IGF1 or the combination of the two.

While Table 17 describes the use of various growth factors at specific concentrations, the invention is not limited to the specific concentrations described herein, as one of skill in the art will recognize that modifying growth factor concentrations is standard in the art and, indeed, is described in the examples above at least by altering activin and heregulin levels and their effects on cell differentiation and organization. Modifications herein include, but are not limited to, concentrations of 5, 10, 20, 50 to 10
0 ng/mL activin; 5, 25, 50 to 75 ng/mL Wnt; 2, 5, 10
, 30 ng/mL heregulin; 10 to 200 ng/mL sonic hedgehog
og(SHH); 10 to 100 ng/mL PDGF; 10 to 20 ng/mL BM
and 25 to 200 ng/mL of IGF1 and/or IGF2. Furthermore, any combination of these factors can be used, and the stage or duration at which they are used can be varied even to a considerable extent without departing from the invention as described.

Example 12
In vivo function of pancreatic endocrine cells To date, enriched populations of endocrine cells generated in vitro from PSCs have not been shown to function in vivo.
We have not generated glucose-responsive insulin-secreting beta cells in vivo.
Applicants tested whether the above method according to Table 17 and described in Example 11 above could generate the first in vitro endocrine cells that are glucose responsive in vivo.

Human pluripotent stem cells were differentiated according to protocol number 3 in Table 17 for stages 1-5, but for stages 6 and 7, in addition to basal DMEM medium with B-27 supplement, F
Only BS, Matrigel and rho kinase inhibitor were used. RNA expression was analyzed by Nanostring at the beginning of stages 5 (d13) and 6 (d15) and at the end of stages 6 (d20) and 7 (d26).
1-D show the relative mRNA expression levels of non-endocrine multipotent pancreatic progenitor subpopulation markers, including PDX1, NKX6.1, SOX9, and PTF1A, and endocrine cell markers, including insulin (INS), glucagon (GCG), somatostatin (SST), and ghrelin (GHRL). At the end of stage 4 (day 13), PDX1, SOX9,
NKX6.1 and PTF1A were all highly expressed. As endocrine differentiation or induction occurs (day 15), these markers all decrease during stage 5. Furthermore, by the end of stage 6 (day 20) or the beginning of stage 7, PDX1, NKX6.1
and PTF1A mRNA levels were decreased, while at the same time, INS, GCG, GHRL
and SST mRNA levels were increased (Fig. 18A-D and Fig. 19A on day 20).
Specifically, PDX1 and PFT1 through stages 5 and 6
The decrease in A expression and the dramatic concomitant increase in the relative mRNA expression of INS, GCG, GHRL and SST are consistent with the upregulation of activin, heregulin and Wnt mRNAs at stages 3 and 4.
The combination of NKX6.1 and gamma secretase (AHW), particularly activin, is responsible for the endocrine differentiation at stages 3 and 4, whereas incorporation of activin at stage 5 induced endocrine differentiation at stages 5, 6, and 7. The similar levels of NKX6.1 expression at days 20 and 26 (stages 6 and 7) compared to the earlier time points shown is consistent with this marker being expressed in pancreatic endocrine cells as well as non-endocrine multipotent pancreatic progenitor subpopulations. Thus, the incorporation of activin at stages 3 and 4 repressed endocrine differentiation at stages 3 and 4, whereas incorporation of gamma secretase at stage 5 induced endocrine differentiation at stages 5, 6, and 7. The similar levels of NKX6.1 expression at days 20 and 26 (stages 6 and 7) compared to the earlier time points shown is consistent with this marker being expressed in pancreatic endocrine cells as well as non-endocrine multipotent pancreatic progenitor subpopulations. Thus, the incorporation of activin at later stages (e.g., stage 5) suppressed endocrine differentiation at stages 3 and 4, whereas incorporation of gamma secretase at stage 5 induced endocrine differentiation at stages 5, 6, and 7.
, 6 and 7) induced the cells towards endocrine differentiation, whereas at the earlier stages, stages 3 and 4, they were inhibited from endocrine differentiation.

After stage 6, the cell aggregates were dissociated and reaggregated (described in more detail in Example 14 below), which removed some cell types generated during stages 3 and 4, including certain PEC subpopulations, e.g., non-endocrine (CHGA-) cells.
The ablation of GA- subpopulation and/or exocrine and/or exocrine progenitor cells and/or ductal and/or ductal progenitor cells can be confirmed by the dramatic decrease in SOX9 and PTF1A after reaggregation (Figure 18, compare days 20 and 26).
After this, the endocrine cells were loaded into the devices on day 26 and implanted into RAG2 mice on day 27, essentially as described above for implantation (or implantation) into SCID beige mice. Figure 20 shows human C-peptide levels in serum one hour (60 minutes) after fasting and glucose administration or challenge. At 10 weeks post-implantation, all five animals were producing insulin, as observed by human serum C-peptide. At 15 weeks post-implantation (Figure 21),
), all animals showed increased insulin production levels compared to levels at 10 weeks post-implantation, with two of the five animals showing an average C-peptide level of 1500 pM, which is consistent with in vivo
This is evidence of robust glucose responsiveness in vo.

Prior to loading the devices with endocrine cells on day 26, samples were taken to verify that functional beta cells had developed, matured, and arisen from the transplanted endocrine cells from stages 6 and 7, for example, and not from any remaining non-endocrine multipotent pancreatic progenitor subpopulation. Immunocytochemistry using both endocrine and non-endocrine markers was performed on the samples. Frozen sections of day 26 samples were co-stained with antibodies using DAPI (FIG. 22A), NKX6.1, and chromogranin A (FIG. 22B). It is clear that the majority of NKX6.1 positive (red) cells also stain positive for the endocrine marker chromogranin (CHGA+). Thus, non-endocrine multipotent pancreatic progenitor cells do not appear to be present in any significant numbers in these endocrine cell preparations, as indicated by the lack of CHGA-/NKX6.1+ cells.

Samples of these same endocrine cell aggregates were also co-stained for INS, NKX6.1 and PDX1 (FIGS. 23A-B).
This is an unexpected result, given that previously published reports of in vitro derived insulin-expressing cells from PSCs did not co-express NKX6.1 and/or PDX1.
No. 7,033,831 to Jiang et al. (2007) and related applications,
Let-like clusters from human embryonic
Stem Cells, Stem Cells 2007 Aug;25(8):1940
See pages 53 to 53 of Epub 2007 May 17.

Taken together, this data provides the first compelling evidence that in vitro PSC-derived endocrine cell cultures (1) can co-express INS, PDX1 and NKX6.1 in vitro and appear glucose responsive in vitro; and (2) can generate glucose-responsive insulin-secreting cells in vivo.

(Example 13)
Retinoic acid, nicotinamide, and BMP upregulate PDX1 and NKX6 in endocrine cells
A hallmark of mature beta cells is that they upregulate NKX6.1 and PDX1 at the individual cell level relative to the levels seen in the non-endocrine (CHGA-) subpopulation of PEC. Examples 11 and 12 show that the majority of cells are in the non-endocrine (CHGA-) subpopulation of PEC.
-) described the generation of endocrine cells with expression levels of NKX6.1 and PDX1 similar to those observed in a subpopulation. It is therefore desirable to identify growth factors that specifically enhance the expression of at least these two markers in insulin-positive endocrine cells.

The two candidate factors are TTNPB, retinoic acid or a retinoid analog, and BM
P4, a member of the bone morphogenetic protein family that is part of the transforming growth factor beta superfamily. TTNPB and BMP4 were tested in various combinations and at various concentrations at stages 6 and 7, typically 0.5 nM and 10 ng/mL, respectively. As shown by Nanostring mRNA analysis in FIG. 24, BMP at stages 6 and 7, or at stage 7 alone, or BMP in combination with TTNPB can simultaneously increase the expression of insulin and PDX1 (FIGS. 24A and C). In contrast, these same stages (stages 6 and 7 and stage 7) were significantly higher in both the aged and healthy subjects, and the expression of insulin and PDX1 was significantly higher in the aged subjects.
TTNPB alone (only BMP) does not appear to increase PDX1 expression compared to BMP alone and even appears to slightly decrease PDX1 expression (Figure 24C, columns 4 and 5).
Compare columns 2 and 3. Under all conditions (BMP alone, TTNPB alone, combination of BMP and TTNPB, each treatment at stages 6 and 7, or stage 7 only), NK
Since NKX6.1 expression is relatively stable, the effect of BMPs on NKX6.1 expression is unclear. As a further indication of BMP specificity, BMPs express differentiation inhibitors (IDs) that are known to be regulated by members of the TGF-β superfamily, including BMP4.
It was also observed that BMP upregulates gene expression (e.g., ID1) (FIG. 24D). Thus, without limiting the present application to any one theory, it is believed that BMP upregulates PDX1 and ID1.
It appears to be a factor that selectively induces the upregulation of

To determine whether any of these markers were co-expressed in any one cell, endocrine cell aggregates were fixed on day 27 of differentiation (after stage 7), cryosections were prepared and stained by immunocytochemistry (ICC) for C-peptide, NKX6.1 and PDX1. See Figures 25-28. Here, instead of staining with INS, cells were stained with an antibody against C-peptide, the central fragment of proinsulin, and thus this staining is also indicative of insulin-expressing cells (Figure 25). The ICC results are shown in Nanostrins, supra.
Consistent with the data in g, BMP alone or in combination with TTNPB at stages 6 and 7, or at stage 7 alone, was able to induce PDX1 expression in insulin (C-peptide)-expressing cells (Figures 25 and 26).

mRNA data show that BMP and TTNPB, alone or in combination, inhibit NKX6.1
Despite the fact that the results suggested that the expression of
Studies have shown that at the cellular level, the co-expression of NKX6.1 and C-peptide (or insulin) expressing cells is quite robust (Figs. 27 and 28), i.e., NKX6.1 staining is bright and often brighter than that observed in non-endocrine multipotent pancreatic progenitor subpopulations. Thus, NKX6.1 expression appears to be largely co-expressed with C-peptide (or insulin) expressing cells. Combined mRNA (Nanostring
) and protein (ICC) data also demonstrate that in certain situations there may be limitations to using only one methodology to initially characterize a cell type.
C Data show that BMP alone or in combination with TTNPB at stages 6 and 7 or at stage 7 alone reduces PDX1 and NKX6 in C-peptide (or insulin) expressing cells.
We demonstrated that it was possible to increase the levels of .1 in the IL-1-binding domain (Figures 27 and 28).
The strongest effect is observed when the two factors are used in combination, but the same is possible with BMP alone. TTNPB may also regulate PDX1 and NKX6.1 independently. For example, TTNPB treatment for fewer days during stage 7, e.g., 2, 3 or 4 days, was able to induce NKX6.1 expression without the presence of BMP (data not shown). Thus, the use of these growth factors may be time-dependent, especially for TTNPB.

With regard to nicotinamide specifically, FIG. 29 shows that nicotinamide in stage 6 culture conditions increases INS and decreases GCG expression, resulting in a three-fold increase in the INS/GCG expression ratio. The function of nicotinamide is unclear, but it is thought to play a role through inhibition of PARP-1, poly(adenosine triphosphate [ADP]-ribose) polymerase-1, and NAD+
Nicotinamide has been reported to prevent beta cell loss in rodents resulting from streptozotocin treatment by blocking their loss. Masiello, P. et al.
98) Experimental NIDDM Development of a N
ｅｗ Model in Adult Rats Administered Store
Ptozotocin and Nicotinamide, Diabetes, 47 (
2): 224-9. Nicotinamide can also increase endocrine differentiation and insulin content in human fetal islets. Otonkoski T. et al. (1993) N
Icotinamide is a potent inducer of endoc
Line differentiation in cultured human
et al panicleatic cells. J Clin Invest 92:1
See pages 459-1466.

Furthermore, mature beta cells express high levels of PDX1 and NKX6.1 in vivo.
o and high levels of PDX1 and NKX6.1 are required for proper function.
Therefore, BMP and BMP combined with TTNPB could potentially inhibit insulin-induced inflammatory responses not only in vivo but also in vivo by inducing high expression of both markers in insulin-positive cells.
We show for the first time that it is possible to generate endocrine cells that are glucose responsive even in vitro.

As noted above, the number of days for stages 6 and 7 is not critical and can be shorter or even longer, with one of skill in the art recognizing this, as the 5 and 10 days for stages 6 and 7, respectively, can be easily manipulated, particularly when using substantially the same growth factors as described herein and according to Table 17.

(Example 14)
Matrigel alone and together with a rho kinase inhibitor enhances cell adhesion of PEC and endocrine cell aggregates. In Example 11 above, cell aggregates were dissociated and reaggregated or reassociated between stages 6 and 7. Dissociation was performed on days 20 or 24, essentially as described in Schulz et al., supra.
On day 0, the cells were aggregated again using Accutase and then rotated.
As described in detail by Fischer et al. (2011), reaggregation removes certain PEC subpopulations, such as any remaining non-endocrine (CHGA-) subpopulations, as well as other non-endocrine cells. Although endocrine cells have an affinity for each other, this interaction is relatively weak in vitro, and even reaggregated cells at stage 6/7 were only loosely associated. Therefore, studies were performed to test various factors and agents for their ability to increase cell-cell interactions within endocrine cell aggregates.

Y-27632, a rho kinase inhibitor, and diluted Matrigel (0.05% v/v)
It was found that the addition of Y-27632 to Matrigel can promote tighter cell junctions in reaggregated endocrine cell aggregates.
At least for example, cell-cell interactions of cell aggregates were increased.

On day 20, differentiation cultures generated substantially as described in Example 12 were cultured in Acc.
The cells were dissociated with utase and reaggregated in db with 5% FBS (lower concentrations of FBS, i.e. 2% or 1% are also effective) and DNase. Y-27632 was not included in the reaggregation medium because doing so would result in the incorporation of the non-endocrine (CHGA-) subpopulation as well as other non-endocrine cells into the newly formed aggregates. The next day and every day thereafter, the cell aggregates were treated with Y-27632 or Matrigel or both components. Stereomicroscope images were taken 1, 2 and 3 days after aggregation as shown in FIG. 30.
Figure 30 demonstrates that the combination of Matrigel, Y-27632 and db-FBS was the most effective. This combination was more effective than the use of db-FBS alone, or db-FBS and Matrigel, or db-FBS and Y-27632 (compare Figures 30A-D). The cell aggregates using the combined agents appeared morphologically tighter than all other samples (Figure 30D). Although it is possible that the agents improve cell-cell contact and cell-substrate interactions, subsequent experiments without FBS and with Matrigel and Y-27632 alone provided the same results. These tighter cell aggregates appear morphologically more organ tissue-like. This is advantageous so that the cell aggregates are better able to withstand the stresses of rolling, packing into devices, processing for implantation, and have improved in vitro and in vivo development and maturation, etc.

(Example 15)
No Noggin Generates PECs Enriched for Non-Endocrine Multipotent Progenitor Subpopulations with Limited Endocrine Cell Content and Without the Need for Fractionation or Purification of the PEC Population As noted above, it is desirable to generate a PEC population (Stage 4) that is highly enriched for non-endocrine multipotent pancreatic progenitor subpopulations (CHGA-), but relatively enriched for cells committed to the endocrine lineage (CHGA+). Such cell populations could be useful, for example, to screen for molecules or conditions that specifically induce their differentiation into endocrine lineage cells. Compounds with this activity could be useful in regenerative medicine applications to generate endocrine cells, including pancreatic beta cells, in vivo or ex vivo.
Because fractionation and purification of specific cell populations from more complex cell mixtures is known to be accompanied by diminishing efficiency due to cell death and other reasons, it would be beneficial to be able to create cell populations highly enriched for a desired phenotype solely through efficient directed differentiation, and thus without the need for a purification step.

Substantially in accordance with Protocol No. 1 of Table 17 for stages 1-4, and in accordance with Schul et al.
As described above, undifferentiated human ESCs were grown and differentiated at stage 3.
The following different conditions were compared over three (3) days in Stage 4: (i) zero Noggin ("No Noggin") for all three days; (ii) 50 ng/mL Noggin for one day and no additional Noggin for two days; (iii) 50 ng/mL Noggin for two days and no additional Noggin for one day; and (iv) 50 ng/mL Noggin for three days. After Stage 3, these conditions (i) to (
Each of the iv) then received standard stage 4 differentiation media cocktails from Tables 8 and 17 without additional Noggin. Flow cytometry analysis after stage 4 demonstrated that no Noggin generated greater than 90% non-endocrine multipotent pancreatic progenitor subpopulations with less than 5% cells committed to the endocrine lineage (CHGA+). See Table 17. Furthermore, each additional day of Noggin treatment generated less non-endocrine multipotent pancreatic progenitor subpopulations (90.6% without Noggin compared to 64.6% with 3 days of Noggin treatment). Conversely, each additional day of Noggin treatment increased the level of cells committed to the endocrine lineage (CHGA+).

QPCR analysis of cell cultures after stage 4 (day 12) showed that at each additional day of Noggin treatment:
We show that there was an increase in the levels of endocrine lineage marker expression, including NKX2.2 and INS (Figures 31B and D), which is consistent with the data from flow cytometry in Table 18. In contrast, expression levels of marker genes for non-endocrine multipotent pancreatic progenitor subpopulations, including NKX6.1 (and PDX1), were relatively abundant at any time point of Noggin treatment.

(Example 16)
Agents that Modulate Expression of Pancreatic Lineage Genes at Stage 7 We next evaluated whether there were any additional modulators of pancreatic lineage gene expression. A differentiation method substantially similar to that described in Examples 13 and 14 was performed (i.e., AHW at stage 3, AH at stage 4, gamma secretase and rho kinase inhibitor at stage 5, matrigel and rho kinase inhibitor at stages 6 and 7). In addition, BMP, Noggin, FGF1, FG1, and IFN-γ were administered at days 30-35 during stage 7.
Other agents, including F2, FGF7, EGF, Hrg, HGF, SCF or TTNPB, were tested for their ability to modulate gene expression. At the end of elongation stage 7 (d35), RNA expression was analyzed by Nanostring.
showed that BMP increased the expression of INS, PDX1 and ID1, consistent with the data shown in FIG. 24 and detailed in Example 13. BMP also decreased GHRL expression. FGF1 (acidic FGF) upregulated GCG and SST. Surprisingly, FGF2 at days 30-35 of stage 7 led to a significant increase in SST expression, accompanied by inhibition or suppression of other hormone genes (e.g., INS, GCG, GH
RL; FIG. 32B, SST panel).

These studies demonstrate that certain agents can still modulate and affect gene expression and therefore cell culture differentiation and the resulting cell types of these more differentiated/committed cells.

(Example 17)
Dissociation and Reaggregation of Cell Cultures Reduces Non-Endocrine (CHGA-) and Increases Appropriately Specified Endocrine (CHGA+) Subpopulations It has previously been noted that dissociation and reaggregation of cell cultures can effectively reduce or reduce the presence of specific subpopulations of cells upon reaggregation, and as a result, reaggregation can be used to enrich for specific subpopulations of cells. Studies were designed to determine the impact, if any, of reaggregated cell cultures with those enriched subpopulations, in this case the endocrine (CHGA+) subpopulation (see Example 14).

The differentiation method was substantially similar to that described in Example 14, including dissociation and reaggregation of hES-derived cell aggregates prior to stage 7 (d20).
A. Nano using a combination of markers including SOX9, PDX1 and NKX6.1
As demonstrated by string analysis, non-endocrine (CHG)
A-) After stage 7 (approximately d29) demonstrating that the subpopulation was significantly reduced (FIG. 33A).
33B shows RNA analysis of INS, GCG, pancreatic polypeptide (P
PY), GHRL, solute carrier family 30 member 8 (SLC30A8), glucose-6-phosphatase 2 (G6PC2), precursor hormone convertase 1 (PCSK
1) and increased endocrine and pancreatic islet cell markers, including glucokinase (GCK).

By themselves, these markers are also observed in other cell types, e.g., PTF1A is expressed in exocrine cells, SOX9 in duct cells, PDX1 and NKX6.1 in beta cells. Therefore, Applicant uses the results of various PE
Data were obtained from endocrine and islet markers. In addition, four PE and non-endocrine (C
HGA-) markers were analyzed (PTF1A, SOX9, PDX1 and NKX6.1)
However, only the PTF1A and SOX9 subpopulations were detected at the end of stage 7 (approximately d25, d26, d
NKX6.1 expression, which is also observed in beta cells and endocrine pancreas, remained high after stage 7. See Figure 33A.

Similarly, other endocrine (e.g., beta and alpha cell) markers, including SLC30A8, G6PC2, PCSK1 and GCK, did not dissociate and reaggregate (or "
Higher expression levels were observed in d20 reaggregated cell cultures compared to "no-reagg" cultures. See FIG. 33C. For example, SLC30A8 is a zinc transporter involved in insulin secretion, and certain alleles of SL30A8 can increase the risk of developing type 2 diabetes; glucose-6-phosphatase 2 (G6PC2) is an enzyme found in pancreatic islets; prohormone convertase 1 (PCSK1), along with PCSK2, processes proinsulin into insulin in pancreatic islets; and finally, glucokinase (GC
GCK) promotes the phosphorylation of glucose to glucose-6-phosphate and is expressed in cells of the liver, pancreas, intestine and brain, where it regulates carbohydrate metabolism by acting as a glucose sensor or by inducing a shift in metabolism or cellular function in response to rising and falling glucose levels, such as occur after a meal or during fasting. Mutations in GCK can cause abnormal forms of diabetes or hypoglycemia. The increased expression of these markers at stage 7 indicates that dissociation and reaggregation of cell cultures effectively reduces or decreases the non-endocrine (CHGA-) subpopulation and increases the endocrine (CHGA+) subpopulation. Thus, applicants have now further refined a method to generate appropriately specified endocrine cells in vitro for in vivo function.

Flow cytometric analysis of stage 1-7 cell cultures demonstrated that dissociation and reaggregation of such cell cultures increased the total endocrine subpopulation (enrichment of CHGA+ cells) and reduced the non-endocrine (
A concomitant reduction in CHGA- or PE subpopulations is also observed.
For more information, please refer to.

To date, functional beta cells can only be differentiated through an in vivo transplantation step, and true functional beta cells in vitro have not yet been described, see Zhou et al. (2008), Nature 455, 627-632.
Thus, for the first time, applicants have demonstrated a properly specified in vitro cell population with greater than 50% endocrine cells (CHIGA+) with less than 10% non-endocrine cells (CHIGA-). Moreover, such cells are capable of developing and maturing in vivo into bona fide functional beta cells and islets; see Example 18 below.

(Example 18)
In vivo insulin production from the endocrine (CHGA+) subpopulation. As a follow-up to Example 17, the enriched endocrine (CHGA+) subpopulation was
A study was designed to determine the in vivo effects of reaggregated cell cultures with the A+) subpopulation.

Human pluripotent stem cell cultures were expanded and differentiated substantially as described in Examples 12-14 and 16 above and in Schulz et al. (2012) supra, except that towards the onset of stage 7 (approximately d20), the cultures were pooled and separated into two samples: 1) cell aggregates were dissociated;
1) were allowed to reaggregate ("reaggregated cell culture"); and 2) cell aggregates that were not dissociated and did not reaggregate (control). In addition, during stage 7, both samples further contained BMP, RA or RA analog, TTNPB, and rho kinase inhibitor and 0.05% Matrigel (cell cultures from d20 to d28). Prior to implanting the samples, samples were taken for Nanostring and flow cytometry analysis (Table 19 above). Table 19 shows that the reaggregated cell cultures around d29 contained approximately 11% more endocrine (CHGA+) subpopulations than the controls. Importantly, the reaggregated samples contained 9.25% fewer non-endocrine (CHGA-) or PE type cells (only 1.45%) than the non-reaggregated samples.

The two samples were separately packed and encapsulated into Applicant's semipermeable, biocompatible ENCAPTRA® EN20™ drug delivery devices (animal-sized study devices) and implanted subcutaneously into a total of 10 SCID beige mice (5 animals received reaggregated cell culture; 5 animals received control cell culture), substantially as described in Example 10 above.

FIG. 34 shows serum human C-peptide levels after fasting (first of three bars from the left for each animal), 30 minutes (30 minutes; second bar) and 1 hour (60 minutes; third bar) after glucose administration or challenge, 21 weeks after implantation or during survival.
After weeks, all but two animals (animals no. 4317 and 4323; one from each cohort) produced robust levels of insulin, mostly as observed by human serum C-peptide ranging from 932 to over 2691 pM. However, when comparing stimulation indices between the two groups (difference in C-peptide serum levels at 30 or 60 min relative to that in fasting serum C-peptide levels), there were no significant differences in in vivo function between the reaggregated cell cultures and the control cohort. Thus, it was demonstrated that the transplanted cultures or grafts were approximately 98% endocrine/CHG-dependent.
Whether composed of A+ reaggregated cell cultures or approximately 87% endocrine/CHGA+ (control), both appear to have in vivo functionality. Thus, Applicants have demonstrated for the first time that in vitro endocrine (CHGA+) cell populations (properly specified endocrine cells) are capable of generating physiologically functional insulin-secreting cells when transplanted.

Thus, for the first time, Applicant has demonstrated that in vitro populations of appropriately specified endocrine cells, when transplanted, develop and mature into functional pancreatic islet cells that secrete insulin in response to blood glucose. Moreover, the in vivo function is similar to that previously described by Applicant for PECs and reported in Schulz et al. (2012) supra. Moreover, as long as appropriately specified endocrine cells are produced by the methods described herein through stages 1-7, additional enrichment of endocrine cells (or loss of non-endocrine cells) by dissociation and reaggregation does not appear to be necessary or required for in vivo function. These aspects of the invention have not been demonstrated heretofore. Previously, Applicant et al. have only demonstrated in vivo function using pancreatic progenitor cell populations and without endocrine cell populations. Indeed, as described in Kelly et al. (2011) supra, PECs have been shown to be capable of producing insulin-secreting, insulin-secreting, and insulin-secreting endocrine cells.
Neither the endocrine (CHGA+) subpopulation nor the pancreatic progenitor preparation generated in vivo function.

(Example 19)
Complex base media for culturing endocrine cells Complex base media such as CMRL and RMPI are widely used to culture islet cells and preserve islet cell mass. CMRL media are generally more complex (more components or ingredients) than RPMI. Furthermore, both CMRL and RMPI are more complex than DMEM, a less complex medium commonly used for cell culture. Thus,
Applicants have investigated whether such a complex base medium might be beneficial in maintaining endocrine cells during later stages of differentiation, such as stages 6 and 7.

Islet transplantation requires the culture and transplantation of islets with sufficient islet cell mass and minimal toxicity. Short-term islet culture is associated with rapid deterioration and reduced viability and glucose control. S. Matsumoto et al. (2003), A comparative example.
Valuation of culture conditions for short
T-term maintenance (<24 hr.) of human islet
s isolated using the Edmonton protocol, C
ell Tissue Banking, 4, 85; and N. J. M. London
(1998), Isolation, Culture and Functional
Evaluation of Islets of Langerhans, Diab
See Etes & Metabolism, 23, pp. 200-207.
A study by Andersson et al. (1978) demonstrated the use of TCM for culturing mouse islets.
The effectiveness of RPMI, CMRL, DMEM and Hams F10 was compared.
rsson A. (1978) Isolated mouse panic
Islets in culture: Effects of serum and
Different culture media on the insulin press
Induction of the islets, Diabetologia, 14, 3
See pages 97-404. Andersson's results showed that Ham's F10 provided the greatest insulin content to the islet cultures, while RMPI provided the best insulin biosynthesis rate to the cultures; this was likely due to the high glucose and nicotinamide in the medium. Still other investigators, such as Davalli et al. (1992), showed that TCM with adenosine phosphate and xanthine was superior to CMRL and RMPI for culturing porcine islets. Davalli, A. M. et al. (1992), In Vitro
function of adult pig islets:Effect of c
Culture in different media, Transplantation
see, 60, 854-860. Davalii et al.
It was further suggested that the high glucose found in I was possibly toxic to pig islets. Thus, islets from different species may have different culture requirements, and there may not be one recipe that is suitable for all endocrine and islet cells.
See, p. 978), p.

Table 20 below lists only the components found in CMRL, RPMI and DMEM media that differ between each media type, i.e., Table 20 does not list components shared by all three types of base media, but only components contained in CMRL but not in RPMI and/or DMEM. Table 20 shows that CMRL is the most complex (highest number of components) and DMEM is the least complex (least number of components). Y indicates the presence of that component in that base media; N indicates the component is not present in the media. In brief, DMEM lacks approximately seven components (N, shaded boxes) that are present in CMRL and RPMI.

The method for generating endocrine cell cultures was substantially similar to that described above, except that stage 6
During the first 10 days of culture, DMEM or CMRL base medium was used for the first 10 days of culture, during the second 10 days of culture, during the third 10 days of culture, during the fourth 10 days of culture, during the fourth 10 days of culture, during the fifth 10 days of culture, during the fifth 10 days of culture, during the sixth 10 days of culture, during the sixth 10 days of culture, during the seventh ...
NC), glucose (Glc) and/or rho kinase inhibitor (Y-27632)
received.

In one experiment, stage 7 cells starting around day 23 were cultured in B27 supplement, Matrigel, B
The cells were cultured in CMRL or DMEM-based media with MP, IGF and Y-27632. Nanostring analysis of d26 (stage 7) samples showed that, in general, stage 7 cells cultured in CMRL had increased non-specific gene expression of hormones and pancreatic endocrine markers compared to cells of the same stage when cultured in DMEM.
See .-C. Thus, CMRL may be important for the maintenance of endocrine cells.

Corresponding studies were also carried out using RPMI compared to DMEM and CMRL-based media (data not shown). CMRL and RMPI media had similar effects on cell culture, both improved over that of DMEM, thus reducing glutathione,
Constituent(s) present in both CMRL and RPMI but not DMEM, including the amino acids hydroxyl-L-proline, L-proline, L-aspartic acid, L-glutamic acid and the vitamins biotin and p-aminobenzoic acid, may be important for the maintenance of endocrine cells during stages 6 and/or 7.

(Example 20)
Cryopreservation does not reduce endocrine cell numbers Commercially viable cell therapy requires scale-up manufacturing of the cell product and a means to store the product long term for on-demand patient needs. Thus, any cell product, whether it be single cells alone or encapsulated cells, such as the ENCAPTRA® drug delivery device, will require cryopreservation or other means for long term storage that does not have deleterious or toxic effects on the cells and does not affect the therapeutic efficacy of the cells, or in this case, the in vivo production of insulin in response to blood glucose. Thus, in ENCAPSULATION OF PANCREATIC CELL, both published on October 2, 2012 and April 23, 2013, respectively,
S DERIEVED FROM HUMAN PLURIPOTENT STEM C
Nos. 8,278,106 and 8,425,922, entitled "LLS"
No. 8; and U.S. Application No. 61/775,480, entitled "Compounds for Polymeric Polymers," filed March 7, 2013.
RYOPRESERVATION, HIBERNATION AND ROOM TER
MPERATURE STORAGE OF ENCAPSULATED PANCRE
ATIC ENDODERM CELL AGGREGATES, and Kroon et al.
Applicants investigated whether cryopreserved stage 7 endocrine cells maintain their ability to develop and function in vivo similar to that observed in fresh (non-cryopreserved) and cryopreserved PECs, as detailed in (2008).

The method for generating endocrine cell cultures was substantially similar to that described above, except that during stage 7, the cell cultures were all in CMRL-based medium and were divided into the following samples: 1)
1) dissociated, not reaggregated, but cryopreserved for a few hours around day 23, then thawed and cultured for several days in CMRL base medium with B27 supplement, BMP, TTNPB and rho kinase inhibitor (no re-agg/no cryopreservation); 2) dissociated, not reaggregated, not cryopreserved (no re-agg/no cryopreservation); 3) dissociated, reaggregated, not cryopreserved (Re-agg/no cryopreservation). All three samples above were cultured in the same stage 7 medium conditions. On day 27, samples were analyzed by flow cytometry. See Table 21 below.

Based on the flow cytometry analysis in Table 21, for cultures that were not dissociated and reaggregated (Samples 1 and 2), cryopreservation of cells (Sample 2) does not reduce the total number of endocrine (CHGA+) cells compared to fresh cells (Sample 1) (88.57 vs. 89.97). As expected, the total endocrine (CHGA+) population is higher (92.85) in cultures that were dissociated and reaggregated and not cryopreserved. Thus, although dissociation and reaggregation affect endocrine cell (CHGA+) numbers as described in Example 17 above, cryopreservation of stage 7 cultures does not appear to change the cellular composition.

Additionally, samples were analyzed using cytoimmunohistochemistry for NKX6.1, C-peptide, INS and GCG staining. See Figures 36A-B. Endocrine (CHGA) from Stage 7
+) Cell aggregates (no re-agg/no cryopreservation; sample 2) showed NKX6.1 (nuclear) and C-peptide (cytoplasmic) co-expression or co-staining (FIGS. 36A-C). Co-staining was not observed in a similar analysis of PECs from the stage 4 differentiation protocol. INS
FIG. 36A-C shows separate staining of GCG (36A; cytoplasmic) and GCG (36B; cytoplasmic) cells.
As can be seen in C, cell cultures from stage 7 were also predominantly monohormonal, i.e.
Most individual cells did not co-express INS and GCG, as seen in the endocrine (CHGA+) subpopulation of PEC from stage 4. Figure 37 shows photomicrographs with a similar staining pattern, but from cell cultures that had been dissociated and reaggregated at the onset of stage 7.

Figure 37 compares graft function from cell cultures 1 and 3 in Table 20 above. Analysis of in vivo function of implanted stage 7 grafts from both cohorts of animals at 12 weeks showed that cultures that were cryopreserved but not reaggregated (sample 1) had similar serum C-peptide levels 30 and 60 minutes after glucose challenge compared to cultures that were not cryopreserved but were reaggregated (sample 3). These C-peptide levels are consistent with the results of the 12 week analysis of cells from Figures 20 and 21.
This is comparable to that observed at weeks 0 and 15 (e.g., at week 15, the mean C-peptide level 60 minutes after glucose challenge was 995 pM). It is therefore anticipated and expected that subsequent analysis will show that in the majority of animals, C-peptide levels will increase to approximately 1000 pM at 30 and/or 60 minutes after glucose stimulation, similar to that described in Example 18 and observed in FIG. 34. Indeed, animal no. 4490, Sample 1 graft, had serum C-peptide levels comparable to animal no. 4320 of Example 18 at 21 weeks post-implantation (FIG. 34).
Peptide levels were obtained at 12 weeks.

Thus, cryopreservation of stage 7 endocrine cells does not appear to affect their in vivo function.

(Example 21)
Increasing glucose concentrations affect hormone expression. Glucose levels in cell culture preparations were increased from 1 g/L (5.5 mM) to 10 g/L (55 mM).
5.5 mM), some of the media are approximately 5.5 mM, which approximates normal blood glucose levels in vivo.
Glucose levels approaching 10 mM are pre-diabetic levels, and 10
Levels above 10 mM resemble a diabetic state. In other words, glucose above 10 mM mimics in vivo hyperglycemic conditions. High glucose can cause secondary post-translational modifications, including, for example, glycation, glyoxidation, and carbonyl stress. Since there are various reports on the effects of high glucose on different cell types in vitro, applicants sought to study the effects of high glucose (i.e., 4.5 g/L or 24.98 mM glucose) on stage 6 and/or 7 endocrine cells.

Glucose is a soluble hexose sugar that is added to all cell culture media, including Ames' medium, Basal Medium Eagle (BME), BGJb medium, Fitton-Jackson modification, Crick's medium, CMRL-1066 medium, and Dulbecco's Modified Eagle's Medium (DMEM).
); DMEM/Ham's Nutrient Mix F-12 (50:50); F-12 Coon's Modification; Fischer's Medium; H-Y Medium (Hybri-Max®); Iscove's Modified Dulbecco's Medium (IMDM); McCoy's Modified 5A Medium; MCDB Medium; Medium 199; Minimum Essential Medium Eagle (EMEM); NCTC Medium; Nutrient Mix, Ham's F-10; Nutrient Mix, Ham's F-12; Nutrient Mix, Ham's F-12 Cain's Modification (F12K);
RPMI-1640; serum-free/protein-free hybridoma medium; Weymouth medium MB
; Williams medium E and various proprietary media. L-15 medium contains galactose instead of glucose. sigmaaldrich.com/life-sci
ence/cell-culture/learning-center/media-
Sigma, available on the World Wide Web at expert/glucose.html.
See a-AldrichMediaExpert.

Media containing glucose supplements at levels greater than 10 mM include at least the following: DMEM/Ham's Nutrient Mix F-12 (50:50) contains 17.5 mM glucose; DMEM (high glucose), GMEM and IMDM all contain 25 mM glucose levels; H-Y Medium (Hybri-Max®) and serum-free/protein-free Hybridoma Medium contain 22.6 and 28.9 mM glucose, respectively. sigmaaldrich.com/life-science/ce
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Sigma-Aldri available on the World Wide Web at
See chMediaExpert. Because different cell culture media contain widely differing levels of glucose and the effects of glucose generally vary between culture systems, the effects of high glucose on endocrine cells were studied.

Again, the method of generating endocrine cell cultures was substantially similar to that described above, except that during stages 6 and/or 7, the cell cultures were treated with exogenous high glucose (total glucose concentration 4.0-5.0 g/ml).
.5 g/L or 25 mM). Thus, this is an additional 3.5 g/L on top of the 1000 mg/L or 1 g/L (5.5 mM) glucose already found in CMRL medium. Both conditions with and without exogenous glucose were treated with the same growth factors including nicotinamide, BMP, RA (or TTNPB), or rho kinase inhibitors and matrigel. Nanostring analysis showed that when high glucose was added at the beginning of stage 6, by the end of stage 6, INS, GCG, and IL-16 were significantly increased.
The results showed that there was an increase in the expression of GHRL and SST, and a decrease in the expression of GHRL (Figure 39A).
and B). Thus, increases in exogenous glucose during stages 6 and 7 increase hormone expression, with the exception of ghrelin.

(Example 22)
Generation of Immature Beta Cells Examples 8-21 describe various iterative methods for the generation of endocrine cells in vitro, including the use of high activin alone or in combination with Wnt and/or heregulin at stage 3, low activin alone or in combination with heregulin at stage 4, noggin and a gamma secretase inhibitor with or without KGF, EGF and a rho kinase inhibitor at stage 5, and one or more of the following nicotinamide, retinoic acid, BMP and matrigel at stages 6-7. Endocrine cell populations generated from such methods are not only monohormonal (e.g., INS only, GCG only or SST only; see FIG. 39), but also co-express other immature endocrine cell markers including NKX6.1 and PDX1.

Flow cytometry analysis was performed on two different stage 7 cultures using INS, SST and GCG hormone staining (Dataset A and Dataset B). One set of data (A) was performed after many repetitions of stages 1-7 had been examined, and the other set of data (B) was performed earlier, e.g., in the absence of nicotinamide, BMP, CMRL, etc. See Table 22 below, which shows the overall percentages of INS, SST and GCG positive staining (see the five left columns), total INS, SST and GCG negative staining (see the five right columns) and single hormone staining (see the center column). The hormone staining from these two data sets (A and B) is compared to that obtained from a stage 4 PEC culture that was maintained in culture until about day 26.

In general, the total INS positive subpopulation (single INS positive plus INS/SST simultaneous positive plus I
The combined NS/GCG co-positivity plus INS/SST/GCG co-positivity was three times greater in stage 7 endocrine cultures than in stage 4 PEC cultures (49.3 vs. 22.7 vs. 15.8).
Accordingly, the total INS alone (without co-expression with SST or GCG) subpopulation was also higher in stage 7 endocrine cultures than in stage 4 PEC cultures (22.7 vs. 12.9 vs. 3.
Importantly, stage 7 cultures were significantly more abundant than stage 4 PEC cultures (3.7/15.8 =
23%) compared with the total INS population (22.7/49.3 = 46% in A and 12.7 in B).
Stage 7 endocrine cell cultures thus appear to consist of more single hormone expressing cells, particularly INS alone expressing cells, than stage 4 PEC cultures. Furthermore, at least Examples 18 and 20 demonstrate that while the still immature stage 7 endocrine cells, when transplanted, develop and mature into physiologically functional islets capable of secreting insulin in response to blood glucose, the endocrine (CHGA+) subpopulation of stage 4 PEC cultures does not secrete insulin in response to blood glucose.
This demonstrates that the same is not possible in vivo. For further details, see Figures 44 and 45 above and the Examples.

(Example 23)
Endocrine (CHGA+) cells from stage 7 and stage 4 (PEC) are distinguishable from each other Although analysis of stage 7 (well-specified endocrine) and stage 4 (PEC) cultures uses the same antibodies (e.g. CHGA, INS, NKX6.1) for cell staining and analysis, the well-specialized endocrine (CHGA+) cell subpopulations from stages 7 and 4 (PEC) are not the same.
Earlier, applicants demonstrated that the origin of functional beta cells observed after transplantation of stage 4 PEC cultures was due to pancreatic precursor or non-endocrine (CHGA-) cells and not primary endocrine (C
Kelly et al. (2011) reported that the expression of PI3K/PEC was not observed in HGA+ cells.
It was demonstrated that endocrine (CHGA+) subpopulations were enriched (purified) in culture and, when transplanted, they did not generate functional islets or beta cells compared to non-enriched PEC cultures.
Kelly et al. (2011), pp. 3-4, FIG. 4 and Appendix 1; Schulz et al. (201
2); and U.S. Pat. Nos. 7,534,608; 7,695,965;
See, for example, US Pat. Nos. 3,920 and 8,278,106. Examples 17, 18 and Table 19 are
We have described a nearly pure (98.2%) endocrine (CHGA+) cell culture from stage 7 that yielded robust functional grafts in vivo when transplanted (FIG. 34). Considering the results of Examples 17, 18 and others described herein, the functional population of stage 7 cultures is endocrine (CHGA+) cells from stage 7, not the significantly smaller percentage of non-endocrine (CHGA-) cells (2.08%). This is in contrast to stage 4 PEC cultures reported in detail by applicants in previous disclosures, where in vivo function is attributed to the non-endocrine (CHGA-) subpopulation. Thus, in general, the endocrine subpopulations from stages 7 and 4 are not comparable or identical.

Table 23 shows flow cytometry data comparing total endocrine (CHGA+) and non-endocrine (CHGA-) subpopulations from stage 7 (well-specified endocrine) and stage 4 (PEC), demonstrating that the subpopulations are not equivalent but distinct from one another. For example, the overall percentage of the CHGA+ subpopulation was significantly higher in stage 7 compared to stage 4 cultures (85.2 vs. 39.4). Accordingly, the overall percentage of the CHGA- subpopulation was significantly lower in stage 7 compared to stage 4 cultures (14.8 vs. 60.1).
Furthermore, true endocrine cells, such as beta cells, not only express CHGA+ but also co-express at least INS and NKX6.1. Thus, true endocrine cells are CHGA+.
GA+/INS+/NKX6.1+ (triple positive) and can function in vivo. Table 23 shows that stage 7 cultures express almost 5 times more CHGA+ than stage 4 cultures.
/INS+/NKX6.1+ (triple positive) cells (37.8 vs. 7.8)
while stage 4 CHGA+/INS+/NKX6.1+ subpopulations may appear to be properly specified as described above, when transplanted, these endocrine subpopulations from stage 4 do not function in vivo, whereas the non-endocrine (CHGA-) subpopulations from stage 4 mature and function in vivo. Thus, Applicants refer to stage 7 endocrine cells as "immature endocrine cells" or "immature beta cells" rather than endocrine precursor/precursor cells, as these cells are developmentally committed to becoming mature beta cells in vivo.

(Example 24)
Purification of endocrine cells from stage 7 cell populations using a zinc sensor Beta cell secretory vesicles contain high concentrations of zinc. Zinc is transported via at least the zinc transporter S
It accumulates in vesicles through the action of LC30A8 (or ZnT8). Zinc-binding fluorescent probes are used to visualize zinc in cells by absorbing and emitting more light at specific wavelengths when bound to zinc than without zinc. So far, reports have shown that
Most probes are unable to localize adequately to detect zinc within beta cell vesicles. Interestingly, PyDPy1 (or Py1; Chemical
Communications, 2011, 47:7107-9) can enter vesicles but has never been used in beta cells.
1 increases the fluorescence intensity by 50-80 fold. For the first time, applicants have demonstrated the generation of endocrine cells or immature beta cell populations capable of in vivo function. Thus, further in
It would be advantageous to have a means to further enrich this population to provide an enriched immature beta cell population for in vitro analysis, for use as a screening tool, or for transplantation.

Stage 7 endocrine cells or immature beta cells were treated with Py1 and the cells were fluorescently sorted for high zinc content.
s custom synthesized by Biopharma, Research Triangle Park, NC) was resuspended in DMSO at 10 mM and diluted with D
The staining solution was diluted in PBS (-/-)/0.25% BSA (buffer A). Differentiated stage 7 cell aggregates were washed twice with DPBS (-/-) and dissociated with Accumax.
Accumax was quenched by the addition of base medium containing -27. The resulting cell suspension was filtered through a 40 micron mesh, centrifuged, washed with Buffer A, and resuspended in staining solution. Staining was continued for 15 min, and the cells were centrifuged, washed with Buffer A, and resuspended in Buffer A for sorting. Cells were sorted by flow cytometry at 4°C and stained using CMRL/
After sorting, cells were centrifuged and resuspended in RNA isolation buffer.

In the results shown in Figure 40, cells surrounded or surrounded by the polygon are live cells with increased fluorescence due to the presence of the Py1 sensor bound to zinc. This cell population was further divided into two approximately equal gates and sorted by their fluorescence intensity into two tubes designated bright and dim. RNA was prepared from the cells and subjected to Nanostring analysis.

As seen in Figures 42A-C, the dim population is composed of INS, IAPP, PDX1, and NKX.
Markers of beta cell lineage such as 6.1, PAX4, PCSK1, G6PC2, GCK and SLC30A8 are enriched. This indicates that the zinc sensor can be used to purify beta cells or immature beta cells from stage 7 cell populations. Previous experiments determined that 630,000 INS mRNA units by Nanostring corresponded to approximately 49% INS positive cells by flow cytometry (data not shown).
Thus, a purified dim population with an INS Nanostring value of 1,200,000 would contain approximately 93% INS-positive cells (1,200,000/630,000 x 4
This would correspond to 9%).

Conversely, the bright sorted population was enriched for markers of the alpha cell lineage, such as GCG, ARX and SLC30A8 (Figures 41, 42, 43).Like beta cells, glucagon cells are also known to take up zinc, and therefore the Py1 sensor can be used to bind zinc in both beta and glucagon cells, but separated or sorted by their different levels of fluorescence.

Summary of Methods for Producing PEC (Stages 1-4) Endocrine Cell (Stages 1-7) Cultures In summary, the invention described herein is directed to at least PEC and immature endocrine cells, and methods for producing such cells including at least stages 1-4 for PEC production and stages 1-7 for endocrine cell production.
FIG. 5 summarizes certain aspects of Applicant's cell compositions and production methods described herein.

Applicant has demonstrated that when transplanted in vivo, endocrine (C
We previously reported that the HGA+ subpopulation does not develop into mature, functioning islet cells.
These endocrine (CHGA)
The non-endocrine (CHGA-) subpopulation of PECs that did not express NGN3 had early or premature NGN3 expression (NGN3 expression prior to PDX1 and KX6.1 co-expression). See also Rukstalis et al. (2009), supra. In contrast, the non-endocrine (CHGA-) subpopulation of PECs that did not express NGN3 had early or premature NGN3 expression (NGN3 expression prior to PDX1 and KX6.1 co-expression).
o develop and mature into endocrine cells.
This delayed NGN3 expression until after in vivo maturation was shown in Example 10 (FIGS. 15-16), where a combination of activin, Wnt and heregulin reduced NGN3 expression compared to controls.
3 and transplantation of these PECs compared to controls improved in vivo function. See Figures 15-16.

Applicants next sought to obtain appropriately specified endocrine cell cultures capable of developing and maturing into pancreatic islets in vivo, and making insulin in response to blood glucose levels similar to what was observed in all cases of PEC, particularly the non-endocrine (CHGA-) subpopulation of PEC. To this end, Applicants performed a number of replicate experiments to suppress or inhibit NGN3 expression at stages 3 and 4. Examples 8-11 detail Applicants' use of activin at various concentrations, alone or together with other agents such as Wnt and heregulin, to affect NGN3 expression or suppression. FIG.
The effects of activin at stages 3 and 4 are also summarized.

Having demonstrated that NGN3 expression can be delayed without affecting in vivo function, Applicants sought to find ways to induce expression of endocrine markers at stages after PEC (stage 4) formation. Examples 11-14 and 16-22 demonstrate in vivo
We describe a number of iterations and methods used to optimize culture conditions to generate appropriately specialized endocrine populations that were capable of generating functional NGNs.
Applicants used a gamma secretase inhibitor at stage 5 to induce CMR 3 expression and endocrine differentiation. Applicants also used CMR 3 expression at stages 6 and 7 to increase endocrine marker expression.
and BMP to increase INS and PDX1. Figures 44 and 45 summarize and describe these efforts.

It will be appreciated that multiple methodologies need to be used initially to characterize and identify the cells (e.g. Q-PCR, ICC, flow cytometry analysis, C-peptide assay, etc.) Q-PCR and Nanostring multiplexed RNA were often used as the only methods to analyze whether such cell types were obtained after fully characterizing and identifying such cells under specific cell differentiation culture conditions.

The methods, compositions and devices described herein are representative of currently preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention. Modifications thereto and other uses that are encompassed by the spirit of the present invention and are defined by the scope of the disclosure will occur to those skilled in the art. Thus, it will be apparent to those skilled in the art that various substitutions and modifications can be made to the present invention disclosed herein without departing from the scope and spirit of the present invention.

As used in the claims below and throughout this disclosure, the phrase "consisting essentially of" is meant to include any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements. Additionally, in embodiments in which numerical values, such as amounts, concentrations, percentages, proportions, or ranges, are recited, it is understood that the value recited may be "at least about" that numerical value, "about" that numerical value, or "at least" that numerical value.

EMBODIMENTS Embodiment 1. In vitro unipotent human immature beta cells.

Embodiment 2. The unipotent human immature beta cell of embodiment 1, which expresses INS and NKX6.1 and does not substantially express NGN3.

Embodiment 3. The unipotent human immature beta cells of embodiment 1, capable of differentiating into mature beta cells.

Embodiment 4. The unipotent human immature beta cell of embodiment 3, wherein said differentiation is in vivo.

Embodiment 5. A unipotent human immature beta cell according to embodiment 1, which forms part of a cell population.

Embodiment 6. Embodiment 5, wherein at least 10% of the cell population are immature beta cells.
2. A unipotent human immature beta cell as described in claim 1.

Embodiment 7. Embodiment 5, wherein at least 20% of the cell population are immature beta cells.
2. A unipotent human immature beta cell as described in claim 1.

Embodiment 8. The method of embodiment 5, wherein at least 30% of the cell population are immature beta cells.
2. A unipotent human immature beta cell as described in claim 1.

Embodiment 9. The method of embodiment 5, wherein at least 40% of the cell population are immature beta cells.
2. A unipotent human immature beta cell as described in claim 1.

Embodiment 10. The unipotent human immature beta cells of embodiment 5, wherein at least 50% of said cell population are immature beta cells.

Embodiment 11. The unipotent human immature beta cells of embodiment 5, wherein at least 60% of said cell population are immature beta cells.

Embodiment 12. The unipotent human immature beta cells of embodiment 5, wherein at least 80% of said cell population are immature beta cells.

Embodiment 13. The unipotent human immature beta cells of embodiment 5, wherein at least 90% of said cell population are immature beta cells.

Embodiment 14. The unipotent human immature beta cells of embodiment 5, wherein at least 98% of said cell population are immature beta cells.

Embodiment 15. The unipotent human immature beta cells of embodiment 1, which are monohormonal.

Embodiment 16. The unipotent human immature beta cell of embodiment 1, which expresses MAFB.

Embodiment 17. A method of generating unipotent human immature beta cells, comprising contacting human definitive endoderm lineage cells in vitro with a TGFβ superfamily member and a Wnt family member, thereby giving rise to immature beta cells.

Embodiment 18. The unipotent human immature beta cells express INS, NKX6.1,
5. The method of embodiment 4, wherein the cell does not substantially express NGN3.

Embodiment 19. The TGFβ superfamily member is Nodal, Activin A,
Activin B, BMP2, BMP4, GDF8, GDF-10, GDF-11 and GD
5. The method of embodiment 4, wherein the medicament is selected from the group consisting of F-15.

Embodiment 20 The method of embodiment 4, further comprising contacting human definitive endoderm lineage cells in vitro with an ERRB receptor kinase activating agent.

Embodiment 21 The method of embodiment 4, wherein the TGFβ superfamily growth factor is activin A.

Embodiment 22 The method of embodiment 4, wherein the Wnt family member is Wnt3a.

Embodiment 23 The method of embodiment 6, wherein the ERBB receptor kinase activating agent is heregulin.

Embodiment 24 The method of embodiment 4, wherein the TGFβ superfamily growth factor is a BMP.

Embodiment 25. The method of embodiment 4, further comprising contacting the human definitive endoderm lineage cells in vitro with nicotinamide, retinoic acid or a retinoic acid analog, a rho kinase inhibitor, or a gamma secretase inhibitor.

Embodiment 26. The method of embodiment 25, wherein the retinoic acid is selected from the group consisting of all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and arotinoid acid (TTNPB).

Embodiment 27 The method of embodiment 26, wherein the retinoic acid is all-trans retinoic acid (RA).

Embodiment 28. The method of embodiment 26, wherein the retinoic acid is 13-cis-retinoic acid (13-cis-RA).

Embodiment 29. The method of embodiment 26, wherein the retinoic acid is arotinoid acid (TTNPB).

Embodiment 30. The rho kinase inhibitor is Y-27632, fasudil, H-1152
26. The method of embodiment 25, wherein the anti-inflammatory agent is selected from the group consisting of P, Wf-536 and Y-30141.

Embodiment 31 The method of embodiment 30, wherein the rho kinase inhibitor is Y-27632.

Embodiment 32. The gamma selenase inhibitor is gamma selenase inhibitor I (GSI
I), Gamma Sectorase Inhibitor II (GSI II), Gamma Sectorase Inhibitor II
I (GSI III), gamma-secretase inhibitor IV (GSI IV), gamma-secretase inhibitor V (GSI V), gamma-secretase inhibitor VI (GSI VI), gamma-secretase inhibitor VII (GSI VII), gamma-secretase inhibitor IX (GSI
I IX), (DAPT), gamma-secreta segregase inhibitor XI (GSI XI), gamma-secreta segregase inhibitor XII (GSI XII), gamma-secreta segregase inhibitor XIII (GS
Gamma-secretase inhibitor XIV (GSI XIV), gamma-secretase inhibitor XVI (GSI XVI), gamma-secretase inhibitor XVII (GSI XVII)
VII), gamma-secretase inhibitor XIX (GSI XIX), gamma-secretase inhibitor XX (GSI XX), gamma-secretase inhibitor XXI (GSI XXI), gamma 40-secretase inhibitor I, gamma 40-secretase inhibitor II, and RO49290.
26. The method of embodiment 25, wherein the compound is selected from the group consisting of:

Embodiment 33. The gamma selenase inhibitor of embodiment 32 is RO4929097.
The method according to

Embodiment 34 The method of embodiment 32, wherein the gamma-secretase inhibitor is GSI IV.

Embodiment 35. A method for generating mature beta cells in vivo, comprising: a.
2. A method comprising: b. contacting human definitive endoderm lineage cells in vitro with a TGFβ superfamily member and a Wnt family member, thereby giving rise to immature beta cells; c. transplanting the immature beta cells of step (a) into a mammalian subject; and c. differentiating the immature beta cells in vivo to generate a population of cells comprising mature beta cells.

Embodiment 36 The method of embodiment 10, further comprising causing said mature beta cells to produce insulin in response to glucose stimulation.

Embodiment 37. The method of embodiment 10, wherein the TGFβ superfamily growth factor is selected from the group consisting of Nodal, activin A and activin B, GDF-8, GDF-11 and GDF-15.

Embodiment 38 The method of embodiment 10, further comprising contacting the human definitive endoderm lineage cells with an ERBB receptor kinase activating agent in vitro.

Embodiment 39. The method of embodiment 10, wherein the TGFβ superfamily growth factor is activin A.

Embodiment 40 The method of embodiment 10, wherein the Wnt family member is Wnt3a.

Embodiment 41 The method of embodiment 37, wherein the ERBB receptor kinase activating agent is heregulin.

Embodiment 42 The method of embodiment 1, wherein the human definitive endoderm lineage cells are contacted with at least 25 ng/mL of a TGFβ superfamily growth factor.

Embodiment 43 The method of embodiment 10, wherein the human definitive endoderm lineage cells are contacted with at least 50 ng/mL of a TGFβ superfamily growth factor.

Embodiment 44 The method of embodiment 10, wherein the human definitive endoderm lineage cells are contacted with at least 75 ng/mL of a TGFβ superfamily growth factor.

Embodiment 45 The method of embodiment 10, wherein the human definitive endoderm lineage cells are contacted with at least 25 ng/mL of a Wnt family member.

Embodiment 46 The method of embodiment 10, wherein the human definitive endoderm lineage cells are contacted with at least 50 ng/mL of a Wnt family member.

Embodiment 47 The method of embodiment 10, wherein the human definitive endoderm lineage cells are contacted with at least 75 ng/mL of a Wnt family member.

Embodiment 48. The method of embodiment 37, wherein the human definitive endoderm lineage cells are contacted with a TGFβ superfamily growth factor and an ERBB receptor kinase activating agent that is 5-15 times less abundant than a Wnt family member.

Embodiment 49. The method of embodiment 10, wherein at least 10% of the cell population are immature beta cells.

Embodiment 50 The method of embodiment 10, wherein at least 20% of said cell population are immature beta cells.

Embodiment 51 The method of embodiment 10, wherein at least 30% of said cell population are immature beta cells.

Embodiment 52 The method of embodiment 10, wherein at least 40% of said cell population are immature beta cells.

Embodiment 53 The method of embodiment 10, wherein at least 50% of said cell population are immature beta cells.

Embodiment 54 The method of embodiment 10, wherein at least 60% of said cell population are immature beta cells.

Embodiment 55 The method of embodiment 10, wherein at least 70% of said cell population are immature beta cells.

Embodiment 56 The method of embodiment 10, wherein at least 80% of said cell population are immature beta cells.

Embodiment 57 The method of embodiment 10, wherein at least 90% of said cell population are immature beta cells.

Embodiment 58. The method of embodiment 10, wherein the immature beta cells express INS and NKX6.1, and do not substantially express NGN3.

Embodiment 59. The immature beta cells express INS, NKX6.1 and MFAB,
11. The method of embodiment 10, which does not substantially express NGN3.

Embodiment 60. A cell line expressing INS and NKX6.1 and substantially no NGN3 expression.
In vitro unipotent human immature beta cells.

Embodiment 61. The unipotent human immature beta cell of embodiment 60, which is capable of maturing into a mature beta cell.

Embodiment 62. The unipotent human immature beta cell of embodiment 60, further expressing MAFB.

Embodiment 63. A method of generating unipotent human immature beta cells, comprising contacting human definitive endoderm lineage cells in vitro with a TGFβ superfamily member and a Wnt family member, thereby generating unipotent human immature beta cells.

Embodiment 64. The unipotent human immature beta cells express INS, NKX6.1,
64. The method of embodiment 63, which does not substantially express NGN3.

Embodiment 65 The method of embodiment 63, further comprising contacting the human definitive endoderm lineage cells with an ERBB receptor kinase activating agent in vitro.

Embodiment 66 The method of embodiment 63, wherein the TGFβ superfamily growth factor is activin A.

Embodiment 67 The method of embodiment 63, wherein the Wnt family member is Wnt3a.

Embodiment 68 The method of embodiment 65, wherein the ERBB receptor kinase activating agent is heregulin.

Embodiment 69. A method for generating mature beta cells in vivo, comprising: a.
2. A method comprising: b. contacting human definitive endoderm lineage cells in vitro with a TGFβ superfamily member and a Wnt family member, thereby giving rise to immature beta cells; c. transplanting the immature beta cells of step (a) into a mammalian subject; and c. maturing the immature beta cells in vivo to generate a population of cells comprising insulin-secreting beta cells.

Embodiment 70 The method of embodiment 69, further comprising contacting the human definitive endoderm lineage cells with an ERBB receptor kinase activating agent in vitro.

Embodiment 71 The method of embodiment 69, wherein the TGFβ superfamily growth factor is activin A.

Embodiment 72 The method of embodiment 69, wherein the Wnt family member is Wnt3a.

Embodiment 73 The method of embodiment 70, wherein the ERBB receptor kinase activating agent is heregulin.

Embodiment 74 The method of embodiment 69, wherein the human definitive endoderm lineage cells are contacted with at least 50 ng/mL of a TGFβ superfamily growth factor.

Embodiment 75 The method of embodiment 69, wherein the human definitive endoderm lineage cells are contacted with at least 25 ng/mL of a Wnt family member.

Embodiment 76 The method of embodiment 70, wherein the human definitive endoderm lineage cells are contacted with a TGFβ superfamily growth factor and an ERBB receptor kinase activating agent that is 5-15 fold lower than that of a Wnt family member.

Embodiment 77 The method of embodiment 69, wherein the immature beta cells express INS and NKX6.1, and do not substantially express NGN3.

Embodiment 78. The immature beta cells express INS, NKX6.1 and MAFB;
70. The method of embodiment 69, wherein the cell does not substantially express NGN3.

Claims (8)

1. A method for generating unipotent human immature beta cells, comprising contacting human definitive endoderm lineage cells in vitro with a TGFβ superfamily member and a Wnt family member, thereby generating unipotent human immature beta cells , wherein the unipotent human immature beta cells express INS and NKX6.1 and do not substantially express NGN3 . The method of claim 1, further comprising contacting the human definitive endoderm lineage cells with heregulin in vitro. The method of claim 1, wherein the TGFβ superfamily member is activin A. The method of claim 1, wherein the Wnt family member is Wnt3a. The method of claim 2 , wherein the heregulin is heregulin-1, heregulin-2, heregulin-3, or heregulin-4. The method of claim 1, wherein the human definitive endoderm lineage cells are contacted with at least 50 ng/mL of a TGFβ superfamily member. The method of claim 1, wherein the human definitive endoderm lineage cells are contacted with at least 25 ng/mL of a Wnt family member. The method of claim 2 , wherein said human definitive endoderm lineage cells are contacted with said TGFβ superfamily member and said heregulin which is 5-15 times lower than said Wnt family member.